Cd19 mabs

CLL cell apoptosis was measured in duplicates as previously described using the ApoScreen Annexin V Apoptosis Kit [29 (link)]. Briefly, cells were resuspended in 150 μl of Annexin V binding buffer containing 1 μl of Annexin V-PE, 1 μl of 7-aminoactinomycin D (7-AAD) and 1 μl of CD19-mAbs (Southern Biotech, Birmingham, AL) followed by flow cytometry on a MACSQuant flow cytometer (Miltenyi Biotec, San Diego, CA). Flow cytometry analysis was performed using FlowJo software (Tree Star, Ashland, OR). P1446A was provided by Piramal Healthcare Ltd. (Mumbai, India). JNK Inhibitor VIII, SP600125, SB203580, ABT-737 and PD0332991 were obtained from Selleck Chemicals (Houston, TX) and CVT-313—from Santa Cruz Biotechnology (Dallas, TX).

Proliferation was quantified using a colorimetric tetrazolium-based assay. Cells were seeded in a 96 well plate at 5000/well and treated with experimental compounds for 72 h. CellTiter96 AQ_ueous One reagent (Promega) was added and the optical density was measured at 490 nm after 4 h. IC₅₀ was calculated using nonlinear regression with a variable slope.
Apoptosis was quantified as described previously using the ApoScreen Annexin V Apoptosis Kit [2 (link)]. Briefly, cells were suspended in Annexin V binding buffer containing 1 µL of Annexin V and 1 µL 7-aminoactinomycin D (7-AAD) per 100 µL of binding buffer. To identify the B-cell population in primary samples, CD19 mAbs (Southern Biotech) was added at the same dilution. After flow cytometry analysis on a LSRFortessa™ (BD Biosciences), data were analyzed with FlowJo software (Tree Star).
AZD4573 was purchased from ChemieTek; JQ1, AZD8835 and copanlisib were from MedChemExpress; AZD1208 and SGI1776 were from Selleckchem.