Cd38 pe cy7

Whole blood was collected on EDTA, then transferred into BD vacutainer CPT tubes (after removal of anticoagulant solution from CPT tubes) and spun at 2500g for 20 min. The PBMCs were collected and one-tenth were used to isolate RNA. The remaining cells were surface stained on ice using three different panels, including Panel A: CD150 BV-421 (clone A12, BD), CD8 AF 647 (clone RPA-T8), CD20 APC-H7 (clone 2H7, BD), CD3 V500 (clone SP34–2, BD), CD14 Pe-cy7 (clone M5E2, BD); Panel B: CD150 BV421, CD3 V500, CCR7 Pe-Cy7 (clone G043H7, Biolegend), CD8 AF647, CD45RA APC-H7 (clone 5H9, BD); and Panel C: CD150 BV421, IgD BV510 (clone IA6–2, BD), CD38 Pe-Cy7 (clone HB7, BD), CD27 AF647 (clone O323, Biolegend), CD20 APC-H7. Cells were acquired on a MACSQuant®10 flow cytometer (Miltenyi).

ROS were analyzed using Cell ROX Green kit (Life technologies, New York, USA) according to manufacturer’s instructions. For positive control, cells were treated with Tert-butyl hydrogen peroxide (TBHP) (working concentration 200 µM). Briefly, after exposure or sham exposure, 1 million cells in 500 µl were taken for ROS measurement and 2 µl of 2.5 mM Cell ROX solution was added directly to the cells in RPMI and incubated for 45 min in CO₂ incubator with closed caps. After 20 min of incubation antibodies against surface markers were added, specifically, CD45-V450 conjugate (BD biosciences, San Jose, California, USA), CD34-APC conjugate (Myltenyi Biotec, Bergisch Gladbach, Germany) specific for HSPC, CD38-PeCy7 (BD biosciences) conjugate to distinguish population that is enriched for either HSC (CD34+ 38−) or progenitors (CD34+ 38+) and 3 µl of 7-AAD for dead cells staining. Samples were then incubated for another 25 minutes in CO₂ incubator and subsequently analyzed by flow cytometer (BD FACS Canto II). Data were analyzed via BD FACS Diva software. Compensation matrix was created by the compensation wizard in the FACS Diva software after acquisition of single color stained samples and unstained control.

Fresh PBMC were used to sort human plasmablast B cells as described before [12 (link)]. Cells were stained with the following antibody panel: CD3 BV421 (BD Biosciences), CD19 APC (BD Biosciences), CD27 FITC (BD Biosciences), CD38 PE Cy7 (BD Biosciences), CD20 PE (BD Biosciences). Plasmablasts were defined as CD3⁻/CD19⁺/CD27⁺/ CD38⁺/CD20⁻ and sorted on a FACS Jazz sorter in single cell mode into a 96 well plate.

Antibodies for intracellular cytokine staining included: anti-CD3-AF700, CD4-PcPCy5.5, CD8-PECF594, IFNg-FITC, CD14-V500, CD19-V500, TNFα-APC, CD38-PECy7, HLADR-BV605 (BD Biosciences); PD-1-PE, IL-2-BV421 (BioLegend); Live-Dead-AquaViD (LifeTechnologies).
The T regulatory cell/activation panel included: anti-CD3-AF700, CD25-PE, HLA-DR-FITC (BD); CD4-PETxR, CD8-APC AF750, Live-Dead-AquaViD (LifeTechnologies); CD39-PECy7, FOXP3-APC (eBioscience); CD127- PE Cy5.5 (Beckman Coulter); CD38- BV421 (BioLegend).

Commercial antibodies and staining reagents originated from BioLegend: CD45.1-FITC, CD45.2-APC, CD38-PE-Cy7, CD4-PerCP, CD8a-PerCP-Cy5.5, IFNAR1 biotin, and streptavidin conjugated to BV786; from BD Biosciences: CD16/CD32, CD138-BV650, B220-PacBlue, CD95 (APO-1/Fas)-PE; from ThermoFisher Scientific: GL7-A488, Ki67-efluor660 and viability dye fixable live/dead stain eFlour780. The 9D11 hybridoma expressing anti-idiotypic monoclonal IgG1 antibody, clone 9D11 (41 (link)), was kindly provided by Elisabeth Alicot, Boston Children’s Hospital, and was conjugated with iFluor647 succinimidyl ester (AAT Bioquest) in-house.

Peripheral B-cell phenotyping was conducted using flow cytometry. 20 µl of normal serum mix consisting of mouse and goat serum (Dako) were pipetted in each FACS vial with the cell suspension, mixed well, and incubated for 10 minutes at room temperature in the dark. Subsequently, 39.5 µl of an antibody mix made of IgD FITC, CD21 PE, CD5 Per-Cy5.5, CD38 PE-Cy7, IgM APC, CD27 APC-H7, CD19 AmCyan (BD Biosciences, San Jose, California, USA), and CD24 Pacific Blue (EXBIO Praha, Vestec u Prahy, Czech Republic) were pipetted into the vials and mixed well. After another 15 minutes of incubation as described above, lysis buffer (BD Biosciences, San Jose, California USA) was added and the vials were incubated for another 10 minutes. After centrifugation at 250×g for 5 minutes, the supernatant was poured off and cells were suspended in 3 ml of PBS and mixed. After another round of washing the supernatant was discarded and the cell pellet was loosened and fixed by adding 250 µl of PBS with 1% formaldehyde. After fixation, measurement was carried out using a FACS Canto II and analyses were performed with BD Facs Diva.

Cultured cells were stained in PBS supplemented with 2% fetal bovine serum (FBS) at 4 °C for 30 min with the following human antibodies: CD34 PE (Biolegend, clone 581, Santiago, CA, US), CD34 FITC (BD Biosciences, clone 581, Franklin Lake, NJ, US), CD38 PE-Cy7 (BD Biosciences, clone HIT2), CD90 APC (BD Biosciences, clone 5E10), CD45 PE (Biolegend, clone HI30), CD11b/Mac1 APC (BD Biosciences, clone ICRF44), CD3 FITC (BD Biosciences, clone HIT3a), CD19 BV201 (Biolegend, clone SJ25C1), anti-mouse CD45 PErCP-Cy5.5 (Biolegend, clone 30-F11) and 7-amino-actinomycin D (7-AAD) (Biolegend). 7-AAD was used to exclude dead cells. Stained cells were washed once with PBS supplemented with 2% FBS and analyzed using the BD LSRFortessa (BD Biosciences). Proportion of positive/negative cells with the same mean fluorescence intensity (MFI) was represented.