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).
Cd8 alexa fluor 700 rpa t8
Manufactured by BD
Sourced in United States
The CD8-Alexa Fluor 700 (RPA-T8) is a fluorescently labeled antibody that targets the CD8 receptor. It is designed for use in flow cytometry applications to identify and analyze CD8-positive cells. The Alexa Fluor 700 dye provides a far-red fluorescent signal that can be detected using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Lsrii flow cytometer, by BD (2 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)
Cd19 bv421 hib19, by BioLegend (1 mentions)
Compbead, by BD (1 mentions)
Quantibrite beads, by BD (1 mentions)
Mid range rainbow fluorescent particles, by BD (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Live dead aqua viability stain, by Thermo Fisher Scientific (1 mentions)
3 protocols using cd8 alexa fluor 700 rpa t8
1
Multiparametric Flow Cytometry Analysis
2
Comprehensive Immunophenotyping of Blood Samples
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EDTA-anticoagulated whole blood samples were stained within 6 h of collection. Briefly, 100 μl of whole blood was stained with an antibody cocktail, diluted in Brilliant Stain Buffer (BD Biosciences). Red blood cells were lysed with FACS lysing solution (BD Biosciences), samples washed and resuspended in FACSflow. Two different antibody panels were used. One panel contained CD8 PerCP (SK1), CD4 FITC (SK3), CD3 APC-H7 (SK7), PD-1 BV786 (EH12.1) and ICOS BV650 (DX29) from BD Biosciences and the other panel contained CD8 Alexa Fluor 700 (RPA-T8), CD4 BV786 (L200), CD3 APC-H7 (SK7), TIGIT APC (MBSA43), CCR5 PE (2D7) from BD Biosciences and HLA-DR PE-Cy5.5 (TU36) from Invitrogen (Waltham, USA). CCR5 density was calculated using BD QuantibriteTM beads (BD Biosciences) [34 (link)]. Samples were acquired on a four laser BD LSRFortessa X-20 (BD Biosciences) within 4 h. CS&T beads and mid-range Rainbow Fluorescent Particles (BD Biosciences) were run before sample acquisition. Compensation was performed for each experiment using BD CompBeads (BD Biosciences). Samples were analyzed using FlowJo software version 9.9.6 (BD Biosciences).
The gating strategy is shown in Supplemental Figure 1A. A representative example of expression of these markers in a mother–infant pair is shown in Supplemental Figure 1B.
The gating strategy is shown in Supplemental Figure 1A. A representative example of expression of these markers in a mother–infant pair is shown in Supplemental Figure 1B.
3
Phenotyping PBMCs by Flow Cytometry
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