CST, 31328S, Human B Cell Signaling Flow Cytometry Panel

Assay Kit for studying CD19/Syk (Tyr525/Tyr526) phosphate/S6 Ribosomal Protein (Ser235/Ser236) phosphate/CD45/Akt1 (Ser473) phosphate/CD79A (Tyr188) phosphate in the research area. Product Usage Information B cell receptor activation can be induced through treatment with Goat Anti-Human IgM, F(ab') 2 Antibody (Low Endotoxin, Azide-free) # 96232 . All antibodies in this kit are compatible with the Intracellular Flow Cytometry Kit (Methanol) # 13593 and can be used in a single staining mix on fixed and permeabilized cells. Prior to fixation and antibody incubation, we recommend adding a fixable viability dye such as the Ghost Dye Violet 510 Fixable Viability Dye # 59863 to enable identification and exclusion of dead cells from analysis. For individual product dilutions, refer to panel product datasheet or individual product pages. Gating strategy for observing B cell receptor activation in B cell populations : Apply the same gating strategy for both untreated and treated cell populations. If a fixable viability dye was used, then first gate on viable cells. Next, gate on CD45+ immune cells. Viewing the CD45+ population, gate on singlets using appropriate scatter parameters, such as FSC-A vs. FSC-H. Observe CD19 vs. SSC-A and gate on CD19+ B cells. Observe phospho-CD79A (Tyr182) and phospho-Syk (Tyr525/526) within the CD19+ gate. Storage CD45 (HI30) Mouse mAb (violetFluor 450 Conjugate) is supplied in 10 mM NaH 2 PO 4 , 150 mM NaCl, 0.09% NaN 3 , 0.1% gelatin, pH 7.2. CD19 (Intracellular Domain) (D4V4B) XP ® Rabbit mAb (Alexa Fluor ® 647 Conjugate), Phospho-CD79A (Tyr182) (D1B9) Rabbit mAb (Alexa Fluor ® 488 Conjugate), and Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb (PE Conjugate) are supplied in PBS (pH 7.2), less than 0.1% sodium azide, and 2 mg/mL BSA. Store at 4 o C. Do not aliquot the antibodies. Protect from light. Do not freeze. All components in this kit are stable in accordance with the date printed on the outer packaging label when stored at the recommended temperature. Please refer to product labels, datasheets, or web pages for specific "Best By" dates for each individual component. Protocol Available protocols: Flow Cytometry (Fixed/Permeabilized) Specificity / Sensitivity Each antibody in the Human B Cell Signaling Flow Cytometry Panel detects endogenous levels of its target protein. CD45 (HI30) Mouse Monoclonal Antibody (violetFluor 450 Conjugate) detects an epitope within the extracellular domain. CD19 (Intracellular Domain) (D4V4B) Rabbit Monoclonal Antibody (Alexa Fluor Species Reactivity: Human Source / Purification Monoclonal antibodies were purified from tissue culture supernatant via affinity chromatography. The purified antibodies were conjugated under optimal conditions, with unreacted dye removed from the preparation.