CD8 T cells were harvested and incubated with HLA-multimers for 15 min at 4 °C and washed three times with cold PBS/BSA prior to staining with antibodies. Cells were incubated with anti-CD3 (clone SK-7, BD), anti-CD4 (clone SK-3, BD), anti-CD8 (clone SK-1, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. T cell activation was measured by intracellular IFNγ staining (XMF1.2, BioLegend) using an intracellular cytokine staining kit (BioLegend) according to manufactures protocol. moDC’s were stained with anti-CD1a (clone HI149, BD), anti-CD14 (clone M5E2, BD), anti-CD80 (clone L307.4, BD), anti-CD83 (clone HB15e, BD), anti-CD86 (clone IT2.2, BioLegend), and anti-HLA-DR (clone G46-6, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. Samples were acquired using a BD LSRFortessa™ flow cytometry machine and analyzed using FlowJo software (Tree Star).
Anti cd8 clone sk1
Manufactured by BD
The Anti-CD8 (clone SK1) is a monoclonal antibody used for the detection and quantification of CD8-positive cells. It is designed for flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 clone ucht1, by BD (2 mentions)
Anti cd27 clone l128, by BD (2 mentions)
Anti cd4 clone l 200, by BD (2 mentions)
Anti cd38 clone ls 198, by Beckman Coulter (2 mentions)
Anti cd45ra clone abb11, by Beckman Coulter (2 mentions)
Anti hla dr clone l243, by BioLegend (2 mentions)
Facs lysing solution, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Intracellular cytokine staining kit, by BioLegend (1 mentions)
Anti cd3 clone sk7, by BD (1 mentions)
Anti cd83 clone hb15e, by BD (1 mentions)
Anti cd86 clone it2, by BioLegend (1 mentions)
Anti hla dr clone g46 6, by BD (1 mentions)
Anti cd14 clone m5e2, by BD (1 mentions)
Anti cd4 clone sk3, by BD (1 mentions)
Lsrii instrument, by BD (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti cd8 clone sk1
1
Characterization of CD8 T cells and moDC's
2
Whole Blood Flow Cytometry Assay
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A whole blood flow cytometry technique was employed as previously described [47 (link)]. Whole-blood samples (200ul) were stained at room temperature for 20 minutes. The following directly conjugated monoclonal antibodies (mAbs) were used: anti-CD3 (clone UCHT1, BD Biosciences), anti-CD4 (clone L-200, BD Biosciences), anti-CD8 (clone SK1, BD Biosciences), anti-CD27 (clone L128, BD Biosciences), anti-CD38 (clone LS-198, Beckman Coulter), anti-CD45RA (clone AbB11, Beckman Coulter) and anti-HLA-DR (clone L243, Biolegend). Red cells were lysed and fixed in FACS lysing solution (BD Biosciences) for 10 minutes in the dark, washed twice in FACS buffer (phosphate-buffered saline (PBS) containing 2% bovine serum albumin and 0.1% NaN3), and fixed with 1% formalin in PBS. Cells were acquired on an LSR-II flow cytometer (BD Biosciences).
3
Whole Blood Flow Cytometry Method
4
PBMC Isolation and T Cell Quantification
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The PBMCs were isolated from whole heparinized blood using density gradient centrifugation [10 (link),11 (link)]. The CD4 and CD8 T cell frequencies were quantified in the PBMCs using directly conjugated monoclonal antibodies. Anti-CD45 (clone D058-1283), anti-CD3 (clone SP34.2), anti-CD4 (clone L200), and anti-CD8 (clone SK1) monoclonal antibodies obtained from BD Biosciences were used for staining. Live/Dead stain (Thermo Fisher Scientific, Waltham, MA, USA) was used to exclude dead cells from the flow analysis. After surface staining and fixation, the cells were acquired on a Becton Dickinson LSRII instrument. At least 50,000 events were acquired from each sample using lymphocyte gating and analyzed using FlowJo software (v10.8, BD Biosciences, Franklin Lakes, NJ, USA). The absolute CD4 count was calculated using complete blood count (CBC) data from hematology and flow cytometry CD4+ T cells percentage analysis.
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