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5 protocols using anti cd3 alexa fluor 700 clone sp34.2

1

Multiparameter Flow Cytometry Assay

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Flow cytometry was performed as described previously (6 (link)). Isolated cells were incubated with predetermined optimized concentrations of anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen), anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen), anti-CD20-APC-Cy7 (clone L27, BD Pharmingen), anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience), anti-CD200-APC (clone OX104, eBioscience), anti-CD4-PerCP (clone L200, BD Pharmingen), anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen), anti-CCR7-APC-Cy7 (clone 3D12, Biolegend), anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend), anti-ICOS-Pacific Blue (C398.4A, Biolegend) and Live/Dead dye-Alexa430 (Invitrogen). After staining the cell surface, cells were permeabilized and fixed by BD perm wash (BD Pharmingen) and washed twice. Finally, cells were incubated with anti-Ki67-FITC (B56, BD Pharmingen), washed twice and diluted in 1% PFA. Data were acquired on a LSRII flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (version 9.2 Tree Star).
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2

Isolation of PBMCs Subpopulations

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From anti-coagulated whole blood DCs, CD4+ T cells and B cells were sorted following staining with monoclonal antibodies to anti-CD45 V450 (clone DO58–1283; BD Pharmingen), anti-CD14 FITC (clone M5E2; BD Pharmingen), HLA-DR PE (clone L243 (G46–6); BD Pharmingen), anti-CD20 ECD (clone B9E9; Beckman Coulter), anti-CD4 PerCP Cy5.5 (clone L200; BD Pharmingen), anti-CD123 APC (clone 7G3; BD Pharmingen), anti-CD11c APC (clone S-HCL-3; BD Pharmingen), anti-CD3 Alexa Fluor 700 (clone SP34.2; BD Pharmingen), anti-CD8 APC.H7 (clone SK1; BD Pharmingen). After staining and washes, stained PBMCs were sorted using the BD FACSAria II. Doublets were excluded from analysis by gating singlets in forward scatter-area (FSC-A) versus forward scatter-height (FSC-H) and side scatter-area (SSC-A) versus side scatter-height (SSC-H) analysis. After gating on lymphocytes (CD45+ followed by FSC-A vs. SSC-A), total B lymphocytes were defined and sorted as CD3- CD8- CD20+ populations. Dendritic cells were gated and sorted as CD3- CD20- CD14- CD11c+/CD123+ cells. Sorted populations were collected into tubes containing RNA Protect (Qiagen) for RNA isolation.
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3

GZMB Expression in Cryopreserved PBMCs

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For Cohort A, cryopreserved PBMCs were thawed at 37°C and washed once in R10 media prior to staining. As samples permitted, 1 to 2 million PBMCs were stained with 2 μl anti-CD3 Alexa Fluor 700 (clone SP34-2; BD Biosciences, San Jose, CA, USA) and 1 μl anti-CD8 Pacific Blue (clone RPA-T8; BD Biosciences) in 150 μl R10 for 30 minutes at room temperature. Cells were then washed with fluorescence-activated cell sorting (FACS) buffer, and fixed with 1% paraformaldehyde. Cells were fixed for 30 minutes at 4°C, and washed once with FACS buffer. Cells were permeabilized by adding 75 μl medium B (Life Technologies, Grand Island, NY, USA), and simultaneously stained for granzyme B with 1 μl anti-GZMB allophycocyanin (clone GB12; Life Technologies) for 30 minutes at room temperature. Cells were washed twice with FACS buffer, fixed in 1% paraformaldehyde, and placed at 4°C until being run on a BD-LSRII (BD Biosciences). Analysis was performed using FlowJo software (version 9.7.1, Tree Star, Ashland, OR, USA). This methodology was then used to assess GZMB expression in Cohort B, with the exception of staining freshly processed cells, and not cryopreserved cells.
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4

CFSE-based SIV Gag Proliferation Assay

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PBMC were stained with CFSE (CellTrace CFSE Cell Proliferation Kit, Invitrogen) and incubated with or without SIVmac239 Gag peptides (2 μg/ml for each peptide). The peptides (obtained through ARRRP) were 15-mers with an 11-amino acid overlap between sequential peptides and represented the complete protein sequence. Cells without any stimuli were used to determine background proliferation. After incubation for 5 days at 37 °C, cells were stained with anti-CD3-Alexa Fluor 700 (clone SP34-2), anti-CD4-PerCP (clone L200), and anti-CD8-PE (clone RPA-T8) Abs (all from BD Pharmingen). After fixation, at least 10,000 CD3+ cells were acquired by flow cytometry, and data were analyzed using FACS-Diva (BD Biosciences) software. The percentages of proliferating CD3+CD4+ and CD3+CD8+ cells were determined by CFSE dilution; background proliferation (without stimulation) was subtracted.
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5

Isolation of PBMCs Subpopulations

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From anti-coagulated whole blood DCs, CD4+ T cells and B cells were sorted following staining with monoclonal antibodies to anti-CD45 V450 (clone DO58–1283; BD Pharmingen), anti-CD14 FITC (clone M5E2; BD Pharmingen), HLA-DR PE (clone L243 (G46–6); BD Pharmingen), anti-CD20 ECD (clone B9E9; Beckman Coulter), anti-CD4 PerCP Cy5.5 (clone L200; BD Pharmingen), anti-CD123 APC (clone 7G3; BD Pharmingen), anti-CD11c APC (clone S-HCL-3; BD Pharmingen), anti-CD3 Alexa Fluor 700 (clone SP34.2; BD Pharmingen), anti-CD8 APC.H7 (clone SK1; BD Pharmingen). After staining and washes, stained PBMCs were sorted using the BD FACSAria II. Doublets were excluded from analysis by gating singlets in forward scatter-area (FSC-A) versus forward scatter-height (FSC-H) and side scatter-area (SSC-A) versus side scatter-height (SSC-H) analysis. After gating on lymphocytes (CD45+ followed by FSC-A vs. SSC-A), total B lymphocytes were defined and sorted as CD3- CD8- CD20+ populations. Dendritic cells were gated and sorted as CD3- CD20- CD14- CD11c+/CD123+ cells. Sorted populations were collected into tubes containing RNA Protect (Qiagen) for RNA isolation.
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