Antibody Validation for Flow Cytometry
Flow cytometry is sensitive, quantifiable, fast, and multiparametric. Large numbers of cells can be analyzed quickly for protein expression, DNA content, cell cycle state, cell size, light scatter characteristics, and ionic shifts—even in very small subpopulations. In addition, modern flow cytometers can measure the intensities of five or more fluorescent markers simultaneously, which is important when the supply of cells is limited. By combining flow cytometry with the highest quality total and activation-state specific antibodies from Cell Signaling Technology, it is possible to examine complex signaling cascades in cell lines, dissociated tissues, aspirates, or hematology specimens. All of our over 550 antibodies validated for flow cytometry, including our fluorochrome-conjugated antibodies, have been screened to determine optimal dilutions and to verify specificity.
Validation Steps Include
- Serial dilution is used to determine optimal dilution.
- Comparison of signal to isotype control is used to estimate the nonspecific binding of primary antibodies.
- Use of known positive and negative cell lines verifies target specificity.
- Treatment of cell lines with pathway-specific inhibitors or activators verifies target specificity.
- The use of blocking peptides, siRNA, and expression vectors verifies specificity of staining.
- Phosphatase treatment confirms phospho-specificity of the antibody.
- Extensive quality control testing guarantees stability over time and eliminates lot-to-lot variability.
- Optimized protocols are provided and dilutions are predetermined.
BrdU (Bu20a) Mouse mAb #5292: Flow cytometric analysis of Jurkat cells, unincorporated (red) or after 30 minutes of BrdU incorporation (blue), using serial dilutions of #5292. The fold-induction ratio is shown in green. Optimal concentration of #5292 was determined to be 0.044 µg/ml.
Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #4324: Flow cytometric analysis of human whole blood, untreated (red) or treated with Human Granulocyte Colony Stimulating Factor (hG-CSF) #8930 (blue), using #4324.
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #4344: Flow cytometric analysis of Jurkat cells treated with U0126 #9903 (blue) or TPA #4174 (green), using #4344.
Positive and Negative Cell Lines
Zap-70 (136F12) Rabbit mAb (Alexa Fluor® 647 Conjugate) #2707: Flow cytometric analysis (A) of Ramos B (blue) and Jurkat T (green) cells using #2707. Two-color flow cytometric analysis (B) of a mixed population of T and B cells (Jurkat and Ramos, respectively) using Zap-70 Antibody and a CD3 antibody. CD3-negative B cells have little or no Zap-70 staining, while CD3-positive T cells stain brightly for Zap-70 protein.