Cell lysates can be prepared utilizing various methods such as reagent-based lysis, physical disruption, or a combination.1
This article outlines an original Quanterix lysate diluent for running lysate samples with Simoa assays diluent. The majority of commonly used ELISA compatible buffers are similarly compatible with the Simoa system. The following are recommendations concerning the lysis buffer when working with cell lysates: 2
- The lysate should be diluted to have less than 0.05% SDS or other detergents that are strongly denaturing when running in an assay. Non-ionic detergents, for example, NP-40 or Triton X-100, are recommended. Zwitterionic detergents, for example, mild ionic detergents such as sodium deoxycholate or CHAPS, are equally compatible. It is recommended to use no more than 2% v/v total detergent.
- The use of sodium azide should be avoided.
- Avoid utilizing greater than 10 mM reducing agents (for example dithiothreitol or mercaptoethanols).
- Lysates should be centrifuged to wash away cellular debris and insoluble fraction. This should be performed for five minutes at 10,000 RCF, or 10 minutes at 5,000 RCF at 4 °C. Save the supernatants.
- Lysates should be aliquoted, snap-frozen, and stored at -80 °C after being centrifuged.
Table 1 outlines a lysis buffer that can be utilized as a generic lysis buffer. It is important to optimize the lysis process according to the assay and cell type. This article presents data employing the Quanterix lysate diluent and samples prepared using this buffer.
Table 1. Recommended cell lysis buffer.
|Tris pH 7–8
|Triton X-100 or NP-40
|Protease inhibitors such as PMSF or cocktail protease inhibitors. Phosphatase inhibitors if relevant
Cell Lysate Simoa Assay Considerations
- Frozen lysates must be entirely thawed, mixed, and centrifuged for five minutes at 20,000 RCF.
- The final assay concentration should be <0.05% after dilution if SDS is utilized in the lysis buffer.
- Lysates can cause the aggregation of Simoa capture beads. This aggregation can result in poor validation data or image analysis errors. Simoa lysate diluents are developed to reduce bead aggregation and keep a high standard of assay performance.
- To minimize bead aggregation further, lysate should be diluted in Simoa Lysate Diluent to have a total protein concentration <1mg/mL when able to do so.
- Protease inhibitors must be added into the lysate diluent. Phosphatase inhibitors should be used where appropriate. Buffers with phosphatase or protease inhibitors must be used straight away. If a buffer has EDTA or EGTA it may alter some inhibitor activity (such as sodium orthovanadate).
Quanterix Lysate Diluents
There are several factors to consider that have an effect on analyte measurement when optimizing an immunoassay. Optimizing the diluents is a critical parameter that impacts sensitivity, measurement accuracy, and specificity.
This article demonstrates the use of Simoa Lysate diluents A, B, C, and D in four assays: HIV p24, programmed death ligand 1 (PD-L1), transforming growth factor alpha (TGFa) and beta (TGFb).
All lysates were assembled employing a Triton-based lysis buffer as described in Table 1. The lysate diluents were tested in a human embryonic kidney (HEK293), HeLa, and Jurkat cell lysates.
Table 2. Description of Simoa lysate diluents.
|Lysate Diluent A
||Tris buffer with bovine serum components and surfactants.
|Lysate Diluent B
||Tris buffer with bovine serum components, high ionic strength, and surfactants.
|Lysate Diluent C
||Tris buffer with bovine serum components, high protein, and surfactants.
|Lysate Diluent D
||Tris buffer with bovine and calf serum components, and surfactants.
|Simoa Lysate Diluent kit
||Lysate Diluent kit containing 30 mL of each diluent (A-D).
Without the lysate sample, lysate diluents were spiked with a calibrator and serially diluted 2x - 64x to evaluate linearity and recovery in four assays known as having measurable signal in infected or normal cell lysates: p24, PD-L1, TGFa, and TGFb.
The unspiked diluent was evaluated for background signal. Three kinds of cell lysate were lysed with Triton RIPA buffer (Table 1) and were tested:
Each lysate was assessed with Lysate Diluents A – D in the four assays. Lysates were prepared to 0.025 mg/mL total protein.
The sample was spiked with the calibrator and then serially diluted 2x – 128x to evaluate linearity and recovery. Along with the dilution linearity, data was investigated for background signal, bead fill %, AEB CV, AEB, concentration CV, and concentration.
Results – Lysate Diluents
The lysate diluents were tested for recovery, background signal, and linearity where lysates were lacking, to understand how the diluents affect the performance of assays.
In p24 and PD-L1 assays, all four lysate diluents demonstrated poor background signal, average dilutional linearity, and average recovery (± 20% of 100% at all tested dilution factors). Lysate diluent C displays a higher non-specific background signal for both TGFa and TGFb (Table 3).
Table 3. Lysate diluent background signal in TGFa and TGFb assays.
|Lysate Diluent A
|Lysate Diluent B
|Lysate Diluent C
|Lysate Diluent D
All diluents’ background signal is above LOQ for TGFb (Table 3). The spike recovery and linearity also appear to be poor. The calibration curves created in lysate diluent have a much steeper slope compared to calibrator diluent (Graph 1).
Graph 1. TGFb calibration curve in lysate diluent vs. calibrator diluent.
TGFb is a good example of optimizing diluents for particular sample types. The TGFb kit is created for use in plasma and serum. Although, when evaluating a lower protein matrix such as cell lysates, the steeper calibration curve in lysate diluent provides a more precise sample reading based on recovery,spike, and dilution linearity.
Results – Cell Lysates
Lysates from three cell lines were tested with all diluents from the four assays. The following sections outline examples for each diluent and how they benefit assay performance with cell lysate samples.
Lysate Diluent A
HeLa cell lysates tested in the TGFb assay demonstrate acceptable dilution linearity in Lysate Diluent A (±20%) up to a 128x dilution. Recovery and spikes are measured at 96.8% at a 2x dilution. The lysates diluted in Lysate Diluent A had a much higher bead fill than lysates diluted in the kit sample diluent (Graph 2).
In addition, diluent A was the sole diluent to display consistent bead fill regardless of lysate concentration (Graph 3).
Graph 2. HeLa cell lysate bead fill in lysate diluent versus sample diluent. Bead fill was normalized as a percentage of average calibrator bead fill.
Graph 3. HeLa cell lysate sample bead fill as a function of dilution factor.
Lysate Diluent B
HEK293 cell lysates analyzed in the TGFa assay present acceptable dilution linearity in Lysate Diluent B (±20%) up to a 128x dilution. Recovery and spike can be measured at 73.5% at a 2x dilution. The bead fill CV’s were extensively decreased in lysate diluent (Graph 4), and bead fill was close to two times higher in lysate diluent than in sample diluent (Graph 5).
Graph 4. Bead fill CV’s in different diluents.
Graph 5. HEK cell lysate bead fill in lysate versus sample diluent.
Lysate Diluent C
Jurkat cell lysates analyzed in the TGFb assay demonstrate acceptable dilution linearity in Lysate Diluent C (±20%) up to a 128x dilution. Recovery and spikes are measured at 94% at a 2x dilution. The bead fill rate in lysate diluent was better than the sample diluent, however, lysates at the same concentration in sample diluent were unavailable for direct comparison (Graph 6).
Graph 6. Jurkat cell lysate bead fill in lysate versus sample diluent.
Lysate Diluent D
When utilized with Jurkat cell lysates in the PD-L1 assay, Lysate Diluent D showed acceptable linearity (within ± 20% of 100%) up to a 128x dilution, and spike and recovery measures at 94.3% at a 2x dilution. Lysates diluted in Lysate Diluent D had a bead fill that could be compared to those diluted in sample diluent (Graph 7).
Graph 7. Jurkat cell lysate bead fill in lysate versus sample diluent.
The Jurkat cell lysates diluted in sample diluent demonstrated less severe bead fill challenges than some of the other assays and cell lines investigated, with bead fill in sample diluent roaming around 75% that of calibrators (Graph 7).
Lysates from three cell lines were tested across four assays with four lysate diluents. Lysate diluents were measured for recovery in the presence and absence of cell lysates, linearity, and background signal. Although not all lysate diluents perform well in all cell lines or assays, each lysate diluent improved assay performance in particular scenarios.
The diluents have shown to positively affect spike or recovery, bead fill, and dilution linearity. These results present the efficacy and demand for the four lysate diluent formulations when used in cell lysate samples measured on the Simoa platform.
References and Further Reading
- Prep Traditional Methods of Cell Lysis https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biologylearning-center/protein-biology-resourcelibrary/pierce-protein-methods/traditionalmethods-cell-lysis.html
- Preparing cell or tissue lysates for ELISA Kits. http://raybiotech.com/blog/preparing-cell-ortissue-lysates-for-elisa-kits/
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