Cd38 pe

Cells were harvested and stained with antibodies at 4°C for 30 minutes, by washing with 1 mL flow cytometry staining buffer, fixed with 1% formalin solution at 4°C for 30 minutes, and resuspended with staining buffer (PBS, 0.5% BSA, and 2 mM EDTA) before analysis. For flow cytometry analysis of ATO cell harvests, a Fc receptor blocking solution, Human TruStain FcX and a fixable viability dye, zombie yellow (BioLegend, San Diego, California) were added. Antibodies (BD Biosciences, San Jose, California) used for cell surface staining were: CD34-APC (581), CD38-PE (HIT2), CD45RA-PE-CF594 (HI100), CD90-BV421 (5E10), CD10-PE-Cy7 (HI10a), CD7-APC-H7 (M-T701), CD5-BV421 (UCHT2), CD1a-FITC (HI149), CD3-FITC or APC (UCHT1), CD4-PE-CF594 (RPA-T4), CD8-PerCP-Cy5.5 (RPA-T8), TCRαβ-FITC (IP26), TCRγδ-BV421 (B1), CD19-PE (HIB19), CD33-PE-Cy7 (WM53), CD56-FITC (B159). Samples analysis was on an LSR4 flow cytometer (BD Biosciences), and data analyzed using the FlowJo software (Tree Star, Washington).

Plasmablasts were isolated from freshly isolated peripheral blood mononuclear cells (PBMCs) collected seven days after Sanaria PfSPZ Vaccine immunization as previously described (Kisalu et al., 2018 (link)). Briefly, PBMCs were stained for viability with Aqua LIVE/DEAD (Thermo Fisher Scientific) followed by surface staining of the following markers: CD3-PE/Cy7 (BD Bioscience), CD19-FITC (BD Bioscience), CD20-APC/Cy7 (BD Bioscience), CD27-APC (Thermo Fisher Scientific), and CD38-PE (BD Bioscience). Plasmablasts were gated as live CD3⁻CD20⁻CD19⁺CD27⁺CD38⁺ and single cell sorted using a BD FACS Aria II (BD Immunocytometry Systems) into 96-well PCR plates containing 20 μL/well of RT reaction buffer from the SuperScript^™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific). Plates were snap frozen on dry ice and stored at −80°C.

For surface staining, cells were stained with surface markers for 30
min on ice and washed twice with FACS buffer (PBS with 2% FBS and 2
mM EDTA buffer). After, cells were fixed and permeabilized with Fix/Perm
buffer (BD Biosciences) for 20 min on ice, washed twice with BD Perm/Wash,
and stained with the intracellular antibodies for 60 min on ice. For PoxP3
staining, cells were fixed and permeabilized with Fix/Perm Buffer
(eBiosciences, San Diego, CA, USA) for 60 min on ice, washed twice with
Perm/Wash buffer (eBiosciences) and stained with intracellular antibodies
for 60 min on ice. Subsequently, the cells were washed twice with the
respective Perm/Wash buffer and kept in 2% paraformaldehyde.
Antibodies used: PBS57-CD1d tet APC (kindly donated by NIH tetramer resource
facility), Vα24 FITC, and CD3 ECD were from Beckman Coulter
(Fullerton, CA), CD4 Qdot655, CD8 Qdot 605, and the viability marker AmCyan
were from Life Technologies (Carlsbad CA, USA), CD25 APC, CD38 PE, HLA-DR
PerCP, IFNγ V450, TNF Alexa700, IL-10 PE, and IL-4 PE-Cy7 were all
from BD bioscience. Data were acquired on a BD LSRFortessa instrument (BD
Biosciences) and analyzed using FlowJo Version 9.8.5 software (TreeStar,
Ashland, OR, USA).