Anti cd19 pacific blue

Single cell suspensions of spleens were prepared by mechanical dissociation of tissue through a 40-μm cell strainer. Red blood cells were then lyzed using ammonium–chloride–potassium lyses buffer (Lonza). Mouse blood leukocytes were collected by lysing red blood cells with ammonium–chloride–potassium buffer and centrifugation at 300g for 5 min. Cells were stained with fluorescent-labeled antimouse antibodies after blocking CD16/CD32 with Fc Block (BD Biosciences). Flow cytometry analysis was performed using following antibodies: Pacific blue anti-CD19, BV421 anti-CD19, APC anti-TCRβ, APC CXCR4, Percp Cy5.5 B220 (all from BioLegend), and PE–anti-CD138 (from BD biosciences). 4′,6-diamidino-2-phenylindole (DAPI) and LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit were purchased from Thermo Fisher Scientific. APRIL-Alexa488 and bovine serum albumin-Alexa488 were prepared by conjugation of APRIL and bovine serum albumin to Alexa Fluor 488 NHS ester (Thermo Fisher). Stained cells or beads were analyzed using a flow cytometer (LSR II; BD), and data were analyzed using FLOWJO, version 10.1, for PC (Tree Star).

Cellular activation was assessed by measurement of HLA-DR and CD38, similar to previous studies (12 (link), 36 (link), 37 (link)). Staining for flow cytometry was performed both extracellularly and intracellularly. The extracellular staining cocktail consisted of LIVE/DEAD Amcyan fixable dye (Thermo Fisher Scientific, Waltham, MA, USA), anti-CD3-APC-H7, anti-CD4-BV605, anti-CD8-BV655, anti-CD14-Pacific blue (all from BD Biosciences, Franklin Lakes, NJ, USA), and anti-CD19-pacific blue (Biolegend, San Diego, CA, USA). The intracellular staining cocktail consisted of anti-CCR5-APC, anti-HLA-DR-PerCP-CY5.5 (all from BD Biosciences, Franklin Lakes, NJ, USA), anti-CD38-PE-CY7 (Biolegend, San Diego, CA, USA) and anti-p24-FITC (Beckman Coulter, Brea, CA, USA). PBMCs were collected at two time-points: day 3 (48 h post stimulation and prior to HIV infection) and day 5 (48 h post infection).

mRNA translation was analyzed using O-propargyl-puromycin (OPP)-labeling and polysome profiling, as described [22 (link)]. For analysis of CLL samples, cells were stained with anti-CD5-PerCyP5.5 and anti-CD19-pacific blue antibodies (BD Biosciences) for 15 min on ice and OPP-labeling was quantified in CD5⁺CD19⁺ cells. For PBMCs from healthy donors, cells were stained with anti-CD5-APC-Cy7, anti-CD19-pacific blue, anti-CD27-PerCP-Cy5.5 and anti-IgG-FITC antibodies (all Biolegend) for 15 min on ice, and OPP-labeling was quantified separately on CD5⁻CD19⁺IgG⁻CD27⁻ and CD5⁻CD19⁺IgG⁻CD27⁺ cells. Polysome profiling was performed as described [22 (link)].

Surface staining of PBMCs was performed with the following flourochrome conjugated monoclonal antibodies: anti-CD19 Pacific Blue, anti-CD10 APC, anti-CD20 APC-Cy7, anti-CD21 FITC, anti-CD27 PE (all Biolegend). Viability Live/Dead yellow dye (Invitrogen) was added to exclude dead cells from analysis. Whenever enough cells were available, corresponding isotype control (Biolegend) staining was also performed. In the event of insufficient cells to run isotype control internal gating controls using markers that provided the cleanest separation between populations were used. Data were acquired on LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo 7.6.4 software (Tree Star, Ashland, OR). Lymphocytes were identified using forward and side scatter, and further gated to include only singlet events and live cells (Figure S1). CD19+ cells were subsequently gated to determine the following B cell subsets: CD10+CD27− immature transitional (IT), CD10−CD21+CD27− naïve, CD10−CD21+CD27+ resting memory (RM), CD20+CD21−CD27+ mature activated (MA) and CD10−CD21−CD27− tissue like memory (TLM). PBMC samples with less than 50% viability on trypan blue stain at the time of thaw or live/dead staining were discarded. 5×10⁵–10⁶ total events were captured. In addition there had to be at least 1000 CD19+ cells to analyze the B cell subsets.