Join our In-Cell ELISA specialist as he discusses an introduction to this exciting application, followed by some useful optimization and troubleshooting tips
In-Cell ELISA, is a well-accepted assay approach to perform quantitative immunocytochemistry of cultured cells using either a colormetric or fluorometric readout. Review the benefits of In-Cell technology, key reagents and optimizing techniques in this webinar.
About the Presenter
John Constable is currently a member of our Scientific Support team. He has a PhD in Physiology from the University of Liverpool, United Kingdom and six years postdoctoral experience at the University of Oregon. His research focused on the development and function of the neuronal synapse.
John spent his graduate studies looking into exocystosis, and his post-doctoral years studying the process of synaptogenesis. He has used a variety of western blotting, immnuofluorescence and ELISA based applications to study these processes.
A really great presentation - great technical specifics"
- Introduction - why use an ICE assay
- Benefits of In-Cell Technology
- Key reagents
- Optimization and Troubleshooting
Hello and thank you for joining us for today's webinar, An Introduction to In Cell ELISA: Principles and Troubleshooting. It is my pleasure to introduce today's presenter, John Constable. John has a PhD in Physiology from the University of Liverpool in the UK, and then six years of postdoctoral experience at the University of Oregon. His research focused on the development and function of neuronal synapse. He spent his graduate studies looking into exocystosis, and his postdoctoral years studying the process of synaptogenesis. He has used a variety of western blotting, immunofluorescence and ELISA-based applications to study these processes.
Tom Ruyle will be joining John today to also discuss In Cell ELISA. Tom has been a scientific support specialist at Abcam for six years. Prior to joining Abcam, Tom spent two years at Columbia University studying ischemia reperfusion injury. Additionally, he has a Bachelor's Degree in Microbiology from the University of Massachusetts. As Nathan highlighted earlier, if you have any questions throughout this presentation we invite you to submit them on the right hand side of your screen on the Q&A panel. Questions will be answered during the troubleshooting portion of this webinar. At this time, I'd like to hand the presentation over to John.
JC: Thank you Sarah and thank you Nathan for that introduction, and thank you to everyone for joining us today. What I hope to achieve today during this introduction to In Cell ELISA webinar, is to give you a basic understanding of what an In Cell ELISA is, why you would want to use one and also how you would go about doing that assay? Just to go over how the presentation overview will flow today, to begin with I'll talk a little bit about what an In Cell ELISA is, why you would want to use one, how it compares to some of the other techniques in the field. We'll then look at a general ICE protocol, and at that the end of that section there will be a video, as Nathan mentioned, and I will prompt you again to turn the speakers on. After that we'll talk about some of the key considerations you need to think about while either using one of our assay kits, or developing your own. A couple of the frequently asked questions we get here in scientific support on this assay, a few tips that will help you along the way and then we'll end with proper resource pages that you may find useful. As both Nathan and Sarah said, we do encourage you to submit questions at any time and we will answer those at the end, as many of those as we can at the end of the presentation, but please do submit them.
So, to be begin with, what I would like to talk about is just what is an In Cell ELISA? For today's purposes, I'll refer to In Cell ELISA either using In Cell ELISA, ICE or even ice; all the same technique. Just to be aware that if you're reading any literature or any other information out there, it is known by several other names in the field and these include In Cell Western, Cell-based ELISA and Quantitative Immunocytochemistry. Now, they are all the same method and they are all a quantitative immunocytochemistry method to measure protein levels, or if you're interested in post-translational modifications, such as phosphorylation, methylation, acetylation, you can use this technique for that. For the purpose of today's talk, the protocol that we go over will be based on how you would use it for cultured adherent cells. You can use it for suspension cells and we will touch on that later in the presentation, but just to be aware that, in general, the protocol that I show you will be specific for the adherent cells.
So that's the introduction to In Cell ELISA. Now, why would you wish to use this technique? Well, one of the big reasons is it's fast and simple. You perform the whole assay in the same wells that you have plated and treated your cells in, so everything's done in one well, there's no transferring of reagents to tubes or anything like that, and you do it all in one plate.
It's accurate and here we see the In Cell ELISA kits freeze the cells at a desired point in time. What that means is if you were to, say, look at the effect of drug treatment over time on your cells and you wanted to fix them at 0, 10, 20, 30, 40 min time points, then what you would be able to do with this assay is get a snapshot of what is happening in the cell at that point in time to your protein or proteins of interest.
It is very specific and all of the kits that we have, the antibodies that are used in this are very highly validated and we make sure that they are only recognizing the protein of interest, and that there is no other cross-reactivity occurring with these antibodies. So you can be very confident in the results that you are getting.
It is a quantitative method and you can use it to produce/reproduce both data, and the data that you do generate can be quantified relative to either total protein levels, or cell numbers. This allows you to compare the results side-by-side, so from one well to another and get an accurate reflection of what is actually happening in the cell at that point in time.
It is a very high throughput assay; traditionally we do these in 96-well plate formats, you can use 384-well plates as well, and so you can normally do a large number of samples at any one time. You can also do a lot of replicates, so you can do it in duplicate or triplicate depending on how much reagent you have. By doing it in those large numbers and in replicates, you're also increasing the accuracy of the results that you're getting, because if you have two or three more wells that basically are giving you the same result, you can be very confident in that result.
The assay is flexible in terms of the readout that you would use to determine a signal, so depending on the reagents available or the equipment, sorry, available in your lab, you can either readout using IR fluorescent dyes or colorimetric detection; and that would be something like horseradish peroxidase or AP, which is the alkaline phosphatase. So depending on what you have available in your lab, this assay can suit both needs.
Finally, you can either do single or dual antibody staining with this technique. An example of why you would want to do dual antibody staining maybe, if you are looking at the phosphorylated stain of a protein in relation to total protein levels. Then you could use this type of assay to look at what's happening to total protein levels versus phosphorylated levels, and so you could use this assay for that type of questions.
That was an introduction to what In Cell ELISA is from the benefits that the technique has. What I would like to do now is just compare it to some of the, another technique that is generally used in the field which is western blotting. Essentially, both of these techniques start off the same way, you would plate the cells in whichever sized plate you were using, you would transfect or treat them with whatever you're interested in doing, and then this is where they would split off. So for western blotting, after you've done the treatment or transfection, you would first lyse the cells, and then you would harvest them and depending on if you were going after a particular fraction or not you may have to do several centrifugation steps after this. You would then quantify the total protein levels in your sample of interest, these samples would then be run on a SDS-PAGE gel, then transferred to either a nitrocellulose or PVDF membrane, depending on what you're using. You would then do the primary and followed by secondary antibody incubations, and finally you would quantify that signal using a densitometry method, depending on what you have available in the lab.
Whereas with In Cell ELISA, after you've done the treatment or transfection of your cells, you would immediately fix them in the same well that they were plated in. You would then do a primary and secondary antibody incubation, and then quantify that signal. So it's only - it's really only a three- or four-step process at this point. Now, if we compare some of the timings that these assays take, and these are just general timings and it really depends on how many samples you have and that, but to lyse your cells for a western blot, you're generally looking at about 30 min or so. It may take about 15 min to harvest the cells and if you have multiple wells that could take a lot longer. Also if you have to do some centrifugation steps, again, that could add some more time. Quantifying the protein, here I've said that's about 1 h but, again, that's really dependent on the method that you're using, and the number of samples that you actually have. To run those samples on the gel, and here it says about 2 h and you could maybe do it a little faster, or a little slower depending on the type of gels you're running, and how fast you run them. Then there's the transfer step, again, here ballpark it's about 1.5 h or so. Again, you could maybe do that a little faster or a little longer, again, depending on your setup. Primary and secondary antibody incubations can be anywhere from 3 or 4 h, again, depending on how good your primary antibody is, you may even need to leave that overnight to get a signal. Then the quantification could - depending on the method you're using - could take about 30 min. So, all in all, if all goes to plan you're looking at about 8-9 h here from starting to lyse your cells to starting to get data.
On the other hand though, with the In Cell ELISA you fix your cells, it's about a 30 min step, and then you proceed with your primary and secondary antibody incubations. Then most of the protocols we have you incubate the primary antibody for either 2 h, or overnight at 4 °C. Really, the overnight at 4 °C step is a convenience factor and that's just in case you start the assay late in the afternoon, you don't have time to stay late that evening so you can just add the primary antibody and pop it in the fridge, and it'll be fine overnight. But you can do the staining in 2 h, and then the primary, the secondary antibody takes about 1 or 2 h. Then, finally, the quantification here takes about 30 min, so, all-in-all, if you do it all in one day you can do this and have results in about 4 h, which is a pretty fast time. But, normally, you're getting the results in 4 h; you're getting a lot of information in 4 h, so if you're running one or two 96-well plates that's a lot of information that you can capture in that timeframe.
So that was an introduction to what In Cell ELISA is, why you would want to use it and how it stacks up against some other commonly-used technique in the field? As we said before, if there's any questions or if there's anything that we haven't explained properly during that last section, please feel free to drop us questions in. What I would like to do now is go on and go through what a general ICE protocol, so just go through it step-by-step and give you an idea of what's happening at each step.
The first slides, what we're looking at here is this is an image of a 96-well plate looking top down, just so you get an idea of what that plate looks like. On the right here, you can see a side on panel of one of those wells.
The first step that you would do in your ICE protocol is to seed the wells into the microplate. At this point, optimal seeding density is very important and you do want the cells to form an even monolayer across the base of the well. You don't want patches everywhere, so the optimal seeding density is very important at this part.
We would then treat the cells as required, so these little orange dots are your treatment or transfection. So depending on what you wanted to do with the cells, you would then do it at this point and this would be entirely dependent on the assay that you're performing. Sometimes you may not need to do this, other times you would, it all depends on the assay.
The third step would be to add the fix to the cells, permeabilize them and then block them with the solutions provided, or with a block that you have yourself.
After that you would then add the primary antibody and, as I mentioned in the previous slide, you can either incubate the primary antibody for 2 h or overnight at 4 °C. Really, in the tests that we've done, there's no significant increase in signal by leaving it overnight compared to 2 h at room temperature. The overnight is really just there for a convenience factor, like I said before, if you don't want to stay late in the lab and you need to get going, you can just pop it in the fridge and deal with it the next day.
You would then add your conjugated secondary antibody, and this would go on for about 1-2 h at room temperature and you really don't want to leave this for more than 2 h at room temperature, because you will start to get some background issues at that point. So this part you want to do when you can finish the assay.
The sixth and final step would be to read the signal, whether it's fluorescent or colorimetric and what I would like to do is at this point, if we look at the stage and say, look, at reading the signal it's different depending on which signal you're going after. So if you're looking at fluorescence and from the majority of our kits when they have fluorescence, you're going to read it at IR700 or 800. You can essentially read these two wavelengths simultaneously on your plate reader, however, when you're doing the colorimetric reaction, so if you're doing dual antibody staining with the AP and HRP, then what you have to do is first add the developer to, say, the AP, the alkaline phosphatase, read that appropriate wavelength. Then after that you would dump off the solution, that solution and then add the developer for the HRP substrate, and then read that wavelength. So if you're doing a colorimetric action there is another step involved there if you're doing dual antibody staining, but it doesn't add too much time. Whether you're using fluorescence or colorimetric signal readings - after both what you would do is you'd Janus Green stain to do a total cell normalization. This is what allows you to be able to compare well-to-well, and get accurate reflections of what's happening.
What I would like to do now is that was a brief run through of what the protocols that you would use for this technique, and what I'd like to do now is show you some of the data that you would capture using one of these assays. What we're looking at here is one of our MitoBiogenesis In Cell ELISA kits, and this may seem a little complicated but hopefully I'll talk you through this pretty easily. If we concentrate on the left panel, first, you can see the top panel in green, what we're looking at is we're looking at COX-1 signal which has been pseudo-colored green. Then below that is the SDH-A panel which has been pseudo-colored red, and underneath that the bottom is a merged image. So where there's intense COX-1 and SDH-A signal, you're going to get yellow in this merge, and where there's predominantly more SDH-A than COX it'll look more orangey.
So there's two things happening in this assay: one is the first six wells, so this side of the plate here is control-treated and this side of the well here has been treated with chloramphenicol. But not only do we have control versus treated cells, we also are looking at the effect on signal between wells that have been plated at 100,000 cells per well all the way down to 1,000 cells per well. So we're capturing two pieces of information here. One thing to note is COX-1 is a mitochondrial encoded protein, whereas SDH-A is actually a nuclear encoded protein and so what we want to do with this assay is look to see what effect chloramphenicol treatment has on these protein levels? I think you can clearly see that in the cells that have been transfected with chloramphenicol, there's a massive decrease in COX-1 signal, but not a SDH-A signal. What this is telling you is that chloramphenicol is specifically inhibiting COX-1 signal, but not SDH-A and also you can see that just as a control you can see that as the number of cells per well decreases, so does the signal intensity, which is what you would expect. So from this assay we can say that chloramphenicol is specifically inhibiting the mitochondrial encoded protein COX-1, but not the nuclear encoded SDH-A. So it's one example of why you would use that to see if a drug treatment is preferentially affecting one protein of interest, but not affecting the cell as a whole, in general. So that's that part.
If we look at this graph here on the right what we can also use this type of assay for is to see, potentially measure the IC50 of a drug's affect. So we're doing the same assay as over here, but what we're doing here is we're testing varying concentrations of chloramphenicol and looking at what increase in the concentration of chloramphenicol added to the wells is doing to relative protein expression levels. For SDH-A, which is this red line, you can clearly see that as the amount of chloramphenicol is increasing it has no effect on this nuclear encoded protein. So the chloramphenicol is not affecting cellular wider processes, because this is our in-house control, our housekeeping gene, if you want to think about it that way. Whereas this green line represents COX-1 relative signal and as the chloramphenicol concentration is added to the wells increased, you get a relative decrease in COX-1 signal. So you can use this to work out the IC50 of the drug's affect, another use of how you could use this kit.
Just to show how reproducible this data is and how accurate it is, what this bottom right panel is showing is the same assay was done in duplicate on three different days. On day one you can see that the first time it was 89% inhibition, the second day you got 85%, so even on the same day there's very little variation between the data you're getting from the kit. The same is true for when they did it on day two, a very small difference between the duplicate values, and the same on day three. On duplicate, very small changes in the number there. So this is normally showing how accurate and reproducible it is, because you do it on the same day, twice in the same day and you get very similar results, but you do day-to-day-to-day and the results are very consistent. So this just shows you how accurate and also how informative this type of assay could be. So that was one type of way you could this assay and so looking at the effect, a drug's effect on one protein level versus another.
Another type of assay you could this for, and I briefly touched on this at the beginning, is to look and see what happens to a phosphorylated form of the protein versus a non-phosphorylated form of the protein. What we're looking at here is this is our Akt total versus Phospho In Cell ELISA kit, and this left hand panel, we're looking at the total Akt-1 signal. We're looking at what happens to that signal as the concentration of PDGF added to the cells is increased. As you can see, the total Akt-1 signal remains pretty constant as the amount of PDGF added to the wells has increased, there's a slight bump up here but nothing dramatic and the standard errors are pretty big there. So not really affecting total Akt-1 levels. However, when we look at what happens to the phosphorylated form of Akt-1, it's pretty obvious here that as you increase PDGF levels you get an increase in the signal of the phosphorylated form of the protein occurring. Again, another example of what this assay is good at measuring.
We're about to show a video clip here, so if you have speakers on your computer I do recommend that you turn them on now. What this video is going to be about, it's looking at one of our other assays which is PARP-1, and we were very lucky recently to have members of JoVE, which is the Journal of Visual Experiments come in and film this assay. I just thought this would be a great way to show you not only the assay in action, but to get a sense of when we say, to wash the plates, how to do that or if we say shake them, how vigorously that you would do that. How to pipette and just generally handle the cells. At the end of it there is a little bit on capturing the data, so you'll get to see some computer shots of the actual data being captured to give you a sense of what that raw data looks like. So I'm just going to start playing the video now, and please turn on your computer speakers and I will be back in a few minutes.
[Start of Video]
In this video the procedure is demonstrated with typical examples of cell line and treatments. However, experimental parameters are defined by the user. To begin, use sterile technique to seed HeLa cells in an amine-coated black-walled, clear bottom 96-well plate at 50,000 cells per well in 50 µl of cell culture medium. Cover the plate and leave it at room temperature for 1 hr. This step will help reduce edge effects, the crescent moon pattern of cells that tend to adhere to the outer edge of wells. After 1 h incubate the plate overnight in a 5% carbon dioxide 37 °C incubator. Next, dilute 4.8 mM staurosporine 200 times in culture media to 24 mM, which is twice the final highest tested concentration. The DMSO concentration in this solution is now 0.5%, and 11 point three-fold dilution series is then prepared using the 24 mM staurosporine and media already supplemented with 0.5% DMSO. As the 12 point include a zero staurosporine control so that all dilutions, including the zero compound control, have the same concentration of the vehicle.
Treat the cells by adding the 12 point staurosporine dilutions at a volume of 50 µl per well in quadruplicate, taking care not to dislodge the cells. The final highest tested staurosporine concentration is 12 mM, and the DMSO concentration in all wells is 0.25%. Place the plate in a 5% carbon dioxide 37 °C incubator for 6 hr. During this portion of the procedure, it is essential that the cells are not left to dry. If using suspension cells the treated cells must be transferred from their original vessel to the provided black-walled, clear bottom 96-well plates in 100 µl of media just prior to the centrifugation. If using adherent cells, as in this demonstration, the black-walled, clear bottom 96-well plate can be put directly in the centrifuge. Centrifuge the plate with cells at 500 x g for 8 min at room temperature. In the fume hood fix the cells by adding 100 µl of 8% paraformaldehyde solution in PBS to each well. Immediately centrifuge the plate with cells at 500 x g for 8 min at room temperature. Then leave the plate in the fume hood and incubate for an additional 15 min at room temperature. This step prevents the loss of cells which are apoptotic and have detached into the media. Gently remove the paraformaldehyde solution and wash each well a total of three times by rinsing with 300 µl of PBS. After removing PBS, permeabilize the cells by adding 200 µl of permeabilization buffer to each well. Then incubate the plate with shaking at room temperature for 30 min. Following careful removal of the permeabilization buffer, add 200 µl of blocking solution to each well. Incubate the plate with shaking at room temperature for 2 hr.
Begin the staining procedure with removal of the blocking solution, followed by the addition of 100 µl of the primary antibody solution to each well in triplicate. Include a background control as described in the written manuscript, cover and incubate the plate with shaking overnight at 4 °C. After overnight incubation, gently remove the primary antibody solution and wash the cells briefly three times by rinsing each well of the plate with 250 µl of wash buffer. This step will remove unbound primary antibody.
Gently remove the last wash buffer and add 100 µl of the secondary antibody solution to each well. Cover the plate and incubate with shaking at room temperature for 2 h, protected from light. Following removal of the secondary antibody solution, wash the cells briefly five times by rinsing each one of the plates with 250 µl of wash buffer. This step will remove unbound secondary antibody. Leave the last wash in the well to prevent the cells from drying. Wipe the bottom of the plate and the reader surface with isopropanol.
Place the plate on the scan surface of the LI-COR Odyssey or Aerius near-infrared imaging system. If using the LI-COR Odyssey imager, set image acquisition parameters as directed in the written protocol accompanying this video. Pre-scan the plate. Wider pixels indicate saturation usually from fibers or other surface contaminants in the 800 channel. To reduce the signal, wipe the plate and reader surface and, if necessary, reduce the 800 channel intensity.
Following the pre-scan, proceed to scan the plate. In the Odyssey application software manually adjust the intensity settings, this will only change the appearance of the scanned image and does not affect the numerical values of integrated intensities. Export and save the plate image. In the Odyssey application software manually add a grid to the analysis. A grid with custom well diameter seven is recommended to capture the entire well surface from each well of a 96-well plate. View the raw values of integrated intensities of the 800 channel in spreadsheet display. Finally, export and save the data.
[End of Video]
JC: Thank you, and I hope you enjoyed that video. Let me just go back to the presentation. Sorry about that, I seem to be having a little issue getting back. Here we go. As I said, I really hope that you enjoyed that video, it's online and this video will be available online at JoVE and, hopefully, there should be a link on the Abcam blog to it very soon. The entire web video is actually about 12/12.5 min long. If you do have time and you're interested in seeing more, I do recommend it because it will go into a little bit more detail in terms of data analysis, and how you would then take that raw data and analyze it in Excel or similar format to generate the graphs that we have looked at. So I do recommend checking it out, and, if anything else, you get to see some of the other people that work here at Abcam, because they make some guest appearances as well. So, yeah, check it out if you can. Now, if any questions came to you during that video or as we went through anything else, please keep sending them in. We will do our best to get to answer as many of them today as we can, and if we don't then myself and everyone else on the tech support team will get to them as soon as possible.
So now what I would like to do is we've talked a little bit about what an In Cell ELISA is, why you would want to use it, how you would do an In Cell ELISA and then we showed the video clip as well. Now what I would like to look at is a few key considerations that you want to keep in mind, some frequently asked questions that we get here in technical support, and a couple of tips that would help you along the way with this type of assay. First what I want to talk about is antibody validation, and I know I touched on this briefly before, but I can't emphasize enough just how important this part is in developing one of these assays. Because you really want to make sure that the signal you're seeing is the correct signal, and so what I'm showing here is this is some of the actual antibody validation data from the Akt, phospho Akt kit that I showed you results for earlier just prior to the video. We have two western blots here: A and B, and western blot A is probed against total Akt-1, whereas western blot B is being probed against the phospho-specific form of Akt-1. They're both loaded equally, so lane one has a recombinant protein, lane two has some non-induced cell extract and lane three has some of that PDGF-induced cell extract.
When we look at membrane A you can clearly see that in both the non-induced versus induced form of cell extract, there's equal amounts of total Akt-1 present, so the induction isn't causing any increase in Akt-1 itself. However, you can clearly see from membrane B that it is causing a huge increase in the phosphorylated form of Akt-1 that is present, because there's no band in lane two, which is the non-induced cell extract.
So not only do we validate these via western blot, we also validate them in immunofluorescence as well. In this panel, this is some vehicle-treated cells and here we have some PDGF-treated cells, and we're staining in these panels total Akt-1, which is shown here pseudo-colored green and Akt-1 phosphorylated serine 473, which is pseudo-colored red. You can see that in the vehicle-treated cells it's predominantly all just total Akt-1, there's very little actual phosphorylated Akt-1 present. However, when you treat the cells with the PDGF you see there's huge induction or increase in the phosphorylated form of the protein. So, really, the take-home message from this is what I've been stressing for, is like only use the most highly validated antibodies in your assay, and be very confident that they are only recognizing your protein of interest.
So one of the most commonly and frequently asked questions we get here in tech support on this is, what density should I seed my cells at? Well, really this is dependent on a few things: (1) is the cell line that you're using, (2) is the abundance of analyte, also the antibody being used, timing of experiment is important to consider here. Really, they all get to the same point, which is at the point that you fix your cells you want to make sure that you have an almost confluent monolayer of cells.
So the factors that you want to consider before doing the experiment is how big are your cells? The smaller they are the more you need to plate, and what their growth rate; if you plate at 9 am one morning, by 9 am the following morning are they going to be completely overgrown? How long you would like to leave your experiment running, so if you plan to do a multi-day treatment, then you should take that into account at the seeding density so that at the point on the final day when you're fixing them they're at that confluent layer. So to be able to do this what we recommend is before you start the In Cell ELISAs, is to run a few dummy plates testing various densities of your cells and see how they grow over time, and then from that you'll be able to find what is the optimal density for your experiment.
One other thing to keep in mind, is that any treatment that you do could affect cell liability and this really creates a problem if the number of cells present and treated versus untreated is very different at the end point. The Janus Green staining can, to a certain extent, correct for this, however, if you have really high cell death or you're assaying really sick cells, then maybe you have to re-evaluate if this is the right way to go about that experiment. Because if you have a lot of cells dying off, then you have to question how reliable and reproducible your results would actually be at that point.
So this one - can I use suspension cells? As I mentioned at the beginning, and it was also mentioned briefly during the movie as well, yes, you can use suspension cells with this protocol, and this is the link to the protocol that you would use for that. Essentially, what you would do is you would culture and treat the cells in one regular 96-well plate, you would then transfer them to a new amine-coated 96-well plate, centrifuge the cells down as is shown in the movie. Add a fixative and then spin again, and then you would proceed as standard with the protocol. So it's pretty straightforward and simple, but this link will give you all that information and a little bit more.
So just a few tips that people find useful and help them with these experiments, and one is which type of plates to use? We recommend the black-walled, clear bottom plates and we do recommend that you use amine-coated plates to help those cells stick down.
When you're pipetting this is the same for pretty much any type of ELISA assay or anything, or even immunofluorescence where you're doing the staining in the well. You really want to avoid touching the pipette tip to the bottom of the well, because what that's going to do is it's normally going to scrape away a lot of your cells, so you lose a lot of signal. You could get some clumps of cells occurring, which is going to also give you some signal and affect your results.
In terms of fixatives, we here generally only use paraformaldehyde during the development phase, and that's how we test them. Other fixatives are possible, so if you wanted to use something like methanol or acetone that shouldn't be a problem. You may want to alter or play with different fixing conditions, so you find the optimal time for that fixative first before you do the ELISA, just because what works for the PFA may not be the same for methanol or acetone.
In terms of controls, you always want to include at least one no primary control well. One's enough, two if you have the room, but at least you want to have one of those.
Also, as I stressed before, really the one of the advantages of this assay is you can run it and replicate it so you can do duplicate or triplicate for every condition, really depending on how much assay or how much sample and kits that you have.
So, hopefully, so far, as I keep saying, we've given you an introduction to In Cell ELISA, some general ICE protocols, a few tips and FAQs that hopefully will help you along the way, and I'm just going to finish off now with a couple of resource pages that you might also find useful.
So here there are some links to the ICE protocols, both on the Abcam and the MitoSciences website. This one down here, the ICE application note is our ICE application guide. It contains a lot of useful information, so if you get a chance scan through it and you'll find it very helpful - at least I do.
This slide I actually - so when I always work in the lab I always find having something like this really useful, especially when you're doing any type of ELISA, or anything like that. Normally, because if you have a 96-well plate and you have multiple treatments going on in different wells, it's sometimes really hard to keep track of what's actually happening. So this will be in the printout that you will be sent, so, as I say, I find it really useful for keeping track of what's happening and what you're doing in every well. Not only that, sometimes you'll collect all the data and you may not get around to analyzing for a day or two, and sometimes you find it hard to remember what you actually did. So having something like this is generally really helpful.
So that is the end of this section of the talk. What I'm going to do now is I'm going to turn you over to Tom, who is also in the scientific support team here at Abcam. What he's going to do is, he's going to talk about some of the other ICE kits that we have in the catalogue and let you know about some special promotions we have ongoing for attending the webinar. During that time what I'm going to do is, I'm going to look through some of the questions that you've been submitting and see which ones we can answer now, and then come back and answer as many as I can in the time we have left. Yeah, I'll be back in a few minutes!
TR: Thank you, John. I hope you've all been enjoying John's presentation so far, and that you've given some thought to how the In Cell ELISA kits might be applicable to your own research. As John goes through your questions and comes up with some answers for your questions, I'll just go through some of the points that John's already made and summarize those. We see, as John mentioned, that the assay kits are extensively validated using relevant cell sample types. We offer assays for a variety of targets relevant to a few fields, including metabolism, cancer and epigenetics. As John mentioned, they are available in colorimetric or near red detection methods and sometimes both, and they're also available either for single protein detection, and in several cases for detection of dual proteins, for instance, the total protein and the phosphorylated protein. The kits have a specificity of western blot analysis, insofar as all of the antibodies are tested by western blotting, and that western blotting data is presented on the datasheets in all cases. Finally, of course, the kits have a sensitivity in quantitative linearity of standard ELISAs.
Currently, we are offering 30 assays and we plan to be adding some more in the future. Here's a list of what we have at the moment, and, as you see, they include a variety of targets applicable in a variety of fields. You'll see we have the PARP-1 cleaved In Cell ELISA kits for apoptosis, several kits that are relevant to response to hypoxia and several kits that are looking at total protein versus phosphorylated protein. So just have a look at those while I have that slide up, and then there's another slide following this one. To generate this list of assays from our website at abcam.com, you would simply type in ‘ELISA’ in the search box and then that results list will include all the In Cell ELISA kits, in addition to all our standard ELISA kits. To filter out the ELISA kits, you can simply click on the link to In Cell ELISA kits to the left of the results list. Of course, you can always look up your protein of interest by typing that into the search box, and that'll bring up all our products, all our primary antibodies, kits relevant to that protein. Here's the remainder of the kits that we're offering at the moment, and you'll see we have kits for phosphorylation of EGFR, total EGFR, JNK, p38 and a variety of other targets.
For the webinar attendees we're offering a promotion up until June 30th, so you'll be able to receive 25% discount on all MitoSciences or metabolism assay kits. To learn more about available products and terms and conditions, simply go to the promotions page and you'll be directed to that promotions page after the end of the webinar. Okay, John, are you ready to take over?
JC: &Yes, thanks Tom, I am ready here and I've been looking at some of the questions that have been submitted during the webinar, sorry. I've been looking through them and I'll try and get through as many as I can in the time that we have left. As I said before, if we don't get to your question today, I do apologize and we will get back to you as soon as we can, so bear with us and hopefully we'll get your response within the next 24 to 48 hr.
So looking through these someone asked: Should there be a wash step after taking the AP substrate reading before adding the HRP substrate? So you'll remember back at the beginning, we showed that if you're doing a colorimetric reaction and you have to do the AP and HRP sequentially. No, you don't need to do any wash step before adding the HRP step, by just dumping off the AP solution and then adding the HRP substrate that is completely fine, there's no additional wash step needed.
Someone asked - let me see, oh, someone did ask: Do I need to have a multichannel pipette for this assay, or will the assay be adversely affected by the extra time needed to treat each well individually? Yes, for this type of assay you do need to have a multichannel pipette, just because it's crucial that the wells do not dry out during this assay, and so having the multichannel pipette will allow you to go a lot faster.
Another technical one - let me see. Oh yeah, someone's asking: Can solutions be poured off, as shown in the video, or should they be aspirated from the plate? The only solution that should be aspirated is PFA, and that's for health and safety reasons. All other solutions can be poured off by upending the plate, as shown in the video, into a suitable waste container.
I think, yes, to answer that one about fixation someone's just, again, asking: Can you use anything other than PFA? So, yes, you can use acetone or methanol.
Someone is asking: You recommend a no primary control well, but is there any other controls necessary and should we do an untreated cell well as well? That's a good question. No, no other controls are necessary, if you have your control versus your treated and then your no primary, that should be more than enough for that assay. So let me have a look.
Someone's asking: If doing double staining do both antibodies have to be from a different species? That's a good question. The antibodies - you can either use a monoclonal and a polyclonal antibody, or if you wish to use two monoclonal antibodies then you would have to make sure that they're different isotypes, so IgG1 versus IgG2a, or IgG2b. So you want to make sure that the secondary antibodies will not cross-react with any of the other primaries. So a good question.
Someone is also asking: Which part of the cell does Janus Green stain? Well, it stains the mitochondria of the cell, and so because of that you'll get a relative number of cells per well, and therefore be able to normalize how many cells you have per well, and normalize your signal intensity from your protein of interest, and so that will allow you to normalize across wells.
Let me see. This one is actually really good, it says: Are these kits' method really quantitative? It's not quantitative in the sense that you can determine an amount of protein per cell or per make, it can be derived so you can't actually put that type of numbers on it. However, the signals are relative between two samples, much in the same way for western blot, but definitely a lot more quantitative than immunocytochemistry or general immunofluorescence. Sorry, I'm just going through these trying to find good general ones.
Someone is asking: Do you need any special software to do the analysis of this data? No, all you need is the software that comes with your spectrophotometer to capture the data, and then you can transfer all that to Excel to create a graph, or any other type of spreadsheet analysis software. This is one part where if you do have a chance to go back and watch the full-length video on the website when we post it, it does a great job of going into a little bit more detail on how to go ahead and analyze that data after you've exported it somewhere like Excel, and so you can capture a little bit more information from that.
Someone - this is a really good question, someone's asking: Can we use other fluorescent secondary antibodies in our kits, in these kits instead of IR dyes, so maybe some of the more traditional DyLights or those type of secondary antibodies? We have not ourselves tested them, and we are kind of doing some in-house testing at the moment. Theoretically, you can use them so if you have a spectrophotometer that can only read certain wavelengths and you can't get up to that IR dye wavelength, then you could use some other secondaries as long as you are completely confident on how specific those are. Just like with the primary antibodies, you would want to run a few controls just showing those secondary antibodies are really just specific to that primary that you're targeting, and there's no cross-reactivity. But if you do those controls, then you should have no issue using other secondaries.
In relation to that, someone asks: Is it possible to do triple staining in the protocol? It's something we ourselves have not tried yet. Theoretically, you can do it if you have really good antibodies in the target, say, one's a polyclonal, one's an IgG1 and one's an IgG2a or 2b, then it's definitely possible that you could do triple staining. You might say you just want to make sure that those antibodies are good, and also that your software can capture all three of those wavelengths that you're looking at.
Let me see. Would you have to do a live dead stain? No, not really, you'd only - that's something you don't want to really consider if the treatment that you were doing to the cells was creating a large amount of die off, in which case you may want to re-evaluate or look at different concentrations of drugs to use. The Janus Green stain is more than enough to normalize the number of cells in your wells. As I say, if you do have to resort to using a live dead stain, maybe you need to, we can do it using a lower concentration of the drug that you're using.
Some general - some people are asking about some of the kits that we have, they want to know how long some of these kits are stable for, even after being opened. These kits are stable for up to six months after being opened, and so that way you can use that a couple of times if you need to and not have to worry about that.
In a similar vein, I think I saw another question somewhere. Here it is: How long can prepared solutions be stored for, such as like any of the solutions that we send with a kit and we do recommend, and this is a thing not only if you use a kit or you're doing this on your own, they should always make up these solutions as fresh as possible. In terms of reading the primaries, we have not tested it but we would not recommend it, just to be on the safe side.
Let me see, I'm just going through and we have a lot more so I'm just trying to find some other ones. Here's one: I see ELISA isn't just suitable for membrane proteins but for intracellular proteins as well? Yeah, it's possible and I think you can do both, depending on what the target is you're going after, but there should be no reason that you couldn't do that.
Someone else is asking: Is it possible to distinguish molecules within organelles in a cell? Using this type of assay, no, just because we're looking at total fluorescence levels rather than zooming in with a microscope and looking at individual compartments within a cell. So for that you would need to use traditional microscopy to look at something like that.
So someone is saying, another protocol question: How does one fix permeabilized in a 96-well plate? Well, hopefully, that one we've entered in the movie that we showed earlier, but essentially you would just pour off the media that the cells are cultured in, add your fixative using the multichannel pipette, leave it for the required period of time, aspirate it off rather than dump the PFA fix and then add your permeabilization.
So I think we're getting to the end, unfortunately, of this webinar. I'm sorry if I didn't get through to all of your questions, I do apologize for that and we will get back to you as soon as possible with all the answers to these; either myself or one of my other colleagues in the scientific support. What I would just like to leave you with is we do have an extensive scientific support team here at Abcam, and we have offices located in the United States; we're in both Boston and Eugene, Oregon. We have an office in Hong Kong and the UK, down in Cambridge, and in Japan. So if you have any other questions about this technique and you haven't had a chance to ask them at the moment, or if you think of anything later please feel free to drop us an email, or give us a call on the information provided.
I just also want to point out that we are having a conference on Stem Cells and Metabolism in November, and there's a link at the bottom of that slide, abcam.com/Metabolism2012, so check it out if you're interested, it should be a great conference. Lastly, I guess I just want to say thank you for attending, it's been great and I hope that during the course of this webinar you've been able to get a good idea of what this technique is, why you'd want to use it and how you would go about doing it. Once again, thank you for joining us today. Bye.