Research settings frequently require the measurement of peripheral blood mononuclear cells (PBMCs) in a complete blood sample. This information can act as an indication of a treatment’s effectiveness or the stage/state of a disease, for instance.
Purifying a blood sample can be expensive, time-intensive and risky as it is possible to lose key samples during the numerous cleaning and purification steps. Manual counting using Trypan Blue demands a dilution and purification step to ensure the sample is manageable due to the considerable amount of erythrocytes and platelets in the sample.
As an alternative, users can skip the PBMC purification procedure when fluorescent counting on the CellDrop™ FL Automated Cell Counter, saving time and money during sample preparation and also boosting count accuracy and reproducibility in comparison to manual counting.
Counting whole blood with AOPI
The CellDrop utilizes a dual-channel fluorescent system to count whole blood samples. Acridine orange (AO) and propidium iodide (PI) disregard extracellular debris and non-nucleated cells, only staining nucleated cells.
Live cells that have been stained by AO will fluoresce green while the dead cells will fluoresce red when stained with PI due to FRET quenching. A combination of the dyes can be added to diluted whole blood in a 1:1 ratio, effectively staining only the cells of interest.
Preparing whole blood for counting on CellDrop
- Dilute whole blood sample 1:100 with PBS. Note: Actual dilution factor is sample dependent and should be established empirically.
- Combine diluted whole blood with AO/PI in a 1:1 ratio for a final dilution of 1:200.
- The sample can then be pipetted into the loading groove of the CellDrop with the arm down.
- Adjust focus and exposure to check the protocol in order to ensure that the appropriate parameters are selected, then press the ‘Count’ button.
Recommended whole blood protocol
CellDrop count algorithms apply a number of parameters, which together are called a count protocol, thus allowing the user to fine-tune what is counted as a cell.
Although some adjustments may still be necessary, the settings recommended for counting PBMCs in whole blood are displayed in Table 1.
Table 1. Recommended Protocol Settings for Whole Blood. Source: DeNovix Inc.
|Green Fluorescence Threshold
|Red Fluorescence Threshold
* Adjust to reflect the actual dilution as required
Ensuring reproducible results
The tips below will offer guidance that ensures the data from whole blood counts on the CellDrop is accurate and consistent:
- To extend the potency of the dyes, store AO/PI in the dark at 4 °C.
- Mix the sample thoroughly in the tube immediately prior to loading.
- Review the focus and fluorescent exposure settings prior to counting and adjust as needed.
- View the counted image after each measurement and adjust parameters as and when required.
Figure 1. CellDrop FL identifies and counts the live and dead nucleated cells in a whole blood sample. Image Credit: DeNovix Inc.
About DeNovix, Inc.
DeNovix Inc. is an instrumentation company that designs, manufactures and sells laboratory equipment to meet the demands of today’s evolving life science technologies. Our focus is on providing innovative products and outstanding customer support. DeNovix is equipped with the financial, commercial and technical resources to deliver breakthrough products for your research success.
DeNovix offers the DS-11 Series Spectrophotometer / Fluorometer, which combines fluorescence analysis and 1uL UV-Vis in the same instrument. Coupled with our new suite of dsDNA Fluorescence assays, DeNovix instruments provide a wider quantification range than any other instrument.
DeNovix instruments are found in life science research labs worldwide. Each instrument is a stand-alone system controlled by a built-in EasyApps™ software. Labs love the smart-phone-like operation, impressive performance and the flexible connectivity of the instrument. Learn more about DeNovix Instruments and how they can benefit your lab.
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