Responder autologous PBMCs were labeled with 5 μM of CellTrace Violet (ThermoFisher) following manufacturer's protocol. 5 × 104 responder cells were plated on 96 round-bottom wells and co-cultured in complete RPMI 1640 medium [Life Technologies] (supplemented with penicillin, streptomycin, L-glutamine [Sigma-Aldrich], and 10% FCS [Natocor]) with unlabeled highly purified CD8+HLA-DR+ or CD8+HLA-DR- T cells in a responder: suppressor ratio of 1:1. Cells were stimulated with 1 μg/mL of plate-coated CD3 (Biolegend) and 1 μg/mL of soluble CD28 (Biolegend). On day 4 post-activation, cells were harvested, and proliferation of CellTrace Violet-labeled cells was assessed by flow cytometry. At this time point, cells were stained with anti-human CD3 FITC (Immunotools), anti-human CD4 BV605 (Biolegend) and anti-human CD8 APC (Immunotools), and incubated with 7-AAD (BD Bioscience) to analyze cytotoxicity and exclude dead cells. Percentage of suppression was calculated by dividing the number of proliferating CellTrace Violet-diluting responder cells in the presence of suppressor cells by the number of proliferating responder cells when cultured alone, multiplied by 100. Unlabeled, stimulated, and unstimulated cells were used as controls.
Anti human cd4 bv605
Manufactured by BioLegend
Anti-human CD4 BV605 is a fluorochrome-conjugated antibody that binds to the CD4 receptor on human cells. It can be used for the identification and enumeration of CD4+ T cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Natocor (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
7 aad, by BD (1 mentions)
Anti human cd38 percp cy5, by BD (1 mentions)
Pe anti human ifn γ, by BioLegend (1 mentions)
Apc anti human cd8, by BioLegend (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Zombie aqua fixable viability kit, by BioLegend (1 mentions)
2 protocols using anti human cd4 bv605
1
Suppressor T Cell Proliferation Assay
2
Activation of SARS-CoV-2-specific T cells
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To minimize measurement errors, freshly isolated PBMCs were preserved in liquid nitrogen until the last follow-up was completed. Upon detection, the PBMCs isolated at the baseline and 4 weeks post the 2nd dose were recovered and washed twice with R10 (RPMI1640 with 10% FBS). After counting, 1 million live (trypan blue negative) cells from each sample were added to 96-well round bottom plates and incubated with either R10 or R10 containing synthesized spike protein peptides (0.6μg/ml for each peptide) overnight. Then, the cells were washed and stained sequentially with live/dead dye(Zombie Aqua Fixable Viability Kit, cat#423101, BioLegend), surface marker antibodies (Anti-human-CD3-APC/cyanine 7, cat#300318, BioLegend; anti-human-CD4-BV605, cat#566148, BioLegend; anti-human-CD8-APC, cat#344722, BioLegend; anti-human-HLA-DR-BV421, cat#307636, BioLegend; anti-human-CD38-Percp-Cy5·5, cat#551400, BD Pharmingen) and an intracellular IFN-γ antibody (anti-human-IFN-γ-PE, cat#502509, BioLegend). Stained samples were analyzed using a BD Fortessa flow cytometer and data were analyzed with FlowJo software version 10 (TreeStar Inc., Ashland, OR, USA). The gating strategy is shown in Supplementary figure 2. The evaluation of T cell activation was based on the data generated from wells incubated with R10.
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