Flow cytometric assays were performed as described previously (11 (link), 12 (link)). Briefly, PBMCs were thawed and co-cultured in a 24-well plate at 1.0 × 106 cells/well in RPMI 1,640 with 10% fetal calf serum for 24 h at 37°C and 5% CO2. After overnight culture, the cells were labeled with the following anti-human monoclonal antibodies: CD3-APC Cy7 (SK7; BD Bioscience), CD4-Alexa700 (RPA-T4; BD Bioscience), CD8-BV480 (PRA-T8, BD Bioscience), CD19-BV421 (HIB19, Bio-Legend), HLA-DR-BV605 (G46-6, BD Bioscience), CD38-BV786 (HIT2, BD Bioscience), PD1-phycoerythrin (PE) (EH12.1, Bio-Legend), CD25-PECy7 (BC96; Bio-Legend), and IL7Ra (CD127)-BV650 (A019D5; Bio-Legend). Isotype controls, 7-AAD, and Fc blocker were included in all experiments. Multicolor flow cytometric analysis was performed using a BD FACS Fortessa analyzer (BD Biosciences) and FlowJo ver. 10 software (Tree Star Inc., Ashland, OR).
Cd19 bv421 hib19
Manufactured by BioLegend
CD19-BV421 (HIB19) is a fluorochrome-conjugated antibody that specifically recognizes the CD19 surface marker. CD19 is a transmembrane protein expressed on B cells and is commonly used as a pan-B cell marker. This antibody can be used for the identification and enumeration of B cells in flow cytometry applications.
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Lab products found in correlation
Cd3 apc cy7 sk7, by BD (1 mentions)
Hla dr bv605 g46 6, by BD (1 mentions)
Cd38 bv786 hit2, by BD (1 mentions)
Cd25 pecy7 bc96, by BioLegend (1 mentions)
Fortessa facs analyzer, by BD (1 mentions)
Cd4 percp cy5, by BD (1 mentions)
Cd69 ecd, by Beckman Coulter (1 mentions)
Ifn γ pe cy7, by BD (1 mentions)
Ccr7 pe, by BioLegend (1 mentions)
Cd8 alexa fluor 700 rpa t8, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
2 protocols using cd19 bv421 hib19
1
Multicolor Flow Cytometry for Immune Cell Analysis
2
Multiparametric Flow Cytometry Analysis
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Following overnight co-culture with stimulator cells, PBMC were harvested, washed in 1× PBS, stained extracellularly, permeabilized, and stained intracellularly, using a 13-color panel as previously described [24 (link), 30 (link), 31 (link)]. The panel used included the following mAbs: CCR7-PE (clone: G043H7, provider: BioLegend), CD4-PerCP-Cy5.5 (L200, BD Biosciences), CD19-BV421 (HIB19, Biolegend), CD3-BV650 (SP34-2, BD Biosciences), integrin α4β7-Alexa Fluor 647 (ACT1, conjugated in house), CD8-Alexa Fluor 700 (RPA-T8, BD Biosciences), CD45RA-APC-Cy7 (5H9, BD Biosciences), CD69-ECD (TP1.55.3, Beckman Coulter), IFN-γ-PE-Cy7 (B27, BD), IL-17A-BV570 (BL168, Biolegend), IL-2-BV605 (MQ1-17H12, BD Biosciences), and TNF-α-BV711 (MAb11, Biolegend). Cell viability was assessed using a Violet Live/Dead viability kit (Invitrogen, Carlsbad, CA, USA). Stained cells were fixed with 1% PFA in PBS. Samples were acquired using a customized LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo v10 (FlowJo, LLC, San Francisco, CA). Responses against IpaB (S. flexneri 2a) were expressed as net percentage of positive cells (i.e., total percentage of positive cells in the presence of IpaB-stimulating cells (targets) minus percentage of positive cells in co-cultures with background stimulators (B-LCL without IpaB).
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