Flow Cytometry Validation
Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002, wortmannin and U0126 (blue), using Phospho-Akt (Ser473) (D9E) Rabbit mAb compared to a nonspecific negative control antibody (red).
Flow cytometric analysis of Jurkat cells, untreated (green), or LY294002/wortmannin/U0126-treated (blue), using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit mAb (Alexa Fluor® 488 Conjugate).
Two-color flow cytometric analysis of asynchronous Jurkat cells using Cyclin B1 (V152) Mouse mAb (Alexa Fluor® 647 Conjugate) #4118 and Phospho-Histone H3 (Ser10) (Alexa Fluor® 488 Conjugate) #9708. Cells represented in green are positive for Cyclin B1 and Phospho-Histone H3, while cells represented in blue are positive for Phospho-Histone H3 and negative for Cyclin B1. Both cell populations (green and blue) correspond to cells undergoing mitosis.
Flow cytometric analysis of Jurkat cells, untreated (left) or anti-CD3 activated (right), using Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody #2701 and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb #9106. Anti-CD3 activation increases the intensity of label with both antibodies.
Flow cytometric analysis of Jurkat cells, U0126-treated (blue) or PMA-treated (green), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370.
Activation State-specific Antibodies Validated 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 activation state-specific antibodies from Cell Signaling Technology (CST), it is now possible to examine complex signaling cascades in cell lines, dissociated tissues, aspirates or hematology specimens.
The analysis of signal transduction is becoming increasingly important in the field of cancer research. Genetic alterations of signaling proteins such as Ras, p53 or receptor tyrosine kinases are involved in most cancers. These alterations induce aberrant downstream signaling, resulting in abnormal gene expression, cell proliferation and cell death in cancer cells. This has led to the development of new anti-cancer drugs such as imatinib that target specific kinase pathways. Using flow cytometry and activation-state-specific antibodies, researchers have examined abnormal signaling pathways in cancer cell lines and patient samples. Furthermore, they have shown that kinase-targeted drugs are effective at reversing the cancer-related abnormal signaling. New flow cytometric-based assays have evolved to examine the efficacy of cancer drugs currently in clinical trials. This research may lead directly to assays that will allow a clinician to identify specific cancer subtypes and treat them with targeted therapy, to monitor the effect of these drugs on patients, and to subsequently monitor residual disease following treatment.
Antibody Validation at CST
All of our flow cytometry-validated antibodies have been screened to determine optimal dilutions and to verify specificity. To determine optimal dilution, an antibody is serially diluted and analyzed on a positive cell line using flow cytometry. The intensity of label at each dilution is compared to that of a similarly diluted nonspecific isotype control. The results are then plotted on a titration curve (mean fluorescence versus dilution) to determine the optimal dilution. In addition, each antibody is tested for activation-state specificity by comparing the label on treated and untreated cells. For example, the label from a phospho-specific antibody should be strong in conditions that are known to favor phosphorylation, and decreased in conditions that inhibit phosphorylation; the label from a total protein antibody should be unaffected by the treatments. Cells may also be treated with phosphatase (CIP-treatment) to confirm the phospho-specificity of the antibody or to estimate the baseline level of phosphorylation. CST also offers a number of blocking peptides that can be used to verify specificity of staining or to examine the level of nonspecific binding.
Antibody Conjugation
CST offers specially formulated fluorochrome-conjugated antibodies. These high-quality conjugates have been optimized for use with flow cytometry to ensure maximal signal with very low background. Extensive quality control testing guarantees stability over time and eliminates lot-to-lot variability. Many of these conjugates are also suitable for immunofluorescent microscopy applications.