Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).
Cd86 it2
Manufactured by BioLegend
Sourced in United Kingdom
CD86 (IT2.2) is a cell surface protein that functions as a co-stimulatory molecule. It is expressed on antigen-presenting cells and provides a co-stimulatory signal necessary for T cell activation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 okt4, by BioLegend (3 mentions)
Pd 1 eh12.2h7, by BioLegend (2 mentions)
Cd14 m5e2, by BioLegend (2 mentions)
Hcd56, by BioLegend (2 mentions)
Cd57 hnk 1, by BioLegend (2 mentions)
Ia6 2, by BioLegend (2 mentions)
Cd20 2h7, by BioLegend (2 mentions)
Cd11c 3, by Thermo Fisher Scientific (2 mentions)
Klrg1 sa231a2, by BioLegend (2 mentions)
Cd8b 2st8.5h7, by Beckman Coulter (2 mentions)
Cd123 6h6, by BioLegend (2 mentions)
Cd16 3g8, by BioLegend (2 mentions)
Hla dr l243, by BioLegend (2 mentions)
Cd19 hib19, by BioLegend (2 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions)
Cd27 m t271, by BioLegend (1 mentions)
Sa231a2, by BioLegend (1 mentions)
Live dead fixable near ir stain, by Thermo Fisher Scientific (1 mentions)
Hit8a, by BioLegend (1 mentions)
Cd80 2d10, by BioLegend (1 mentions)
4 protocols using cd86 it2
1
Flow Cytometric Profiling of PBMCs
2
Multiparametric Flow Cytometry Assay
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Unpurified ascites cells were seeded at 200,000 cells/well in 100 μL medium into flat-bottom low-adherence 96-well plates. After resting overnight, cells were treated with BiTEs/TriTEs or viruses, diluted in 100 μL medium or autologous ascites fluid. Five days later, cells were harvested and processed for flow cytometry, using Live/Dead Fixable Near IR stain (Invitrogen, UK, #L10119) and anti-CD11b (ICRF44, Biolegend, UK, #301310), −CD64 (10.1, Biolegend, UK, #305006), −CD86 (IT2.2, Biolegend, UK, #305422), −CD80 (2D10, Biolegend, UK, #305208), −CD4 (OKT4, Biolegend, UK, #317408), −CD8 (HIT8a, Biolegend, UK, #300912) and -CD25 antibodies (BC96, Biolegend, UK, #302606).
3
Detailed Immunophenotyping of T-cell Subsets
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The following antibodies were used: CD80 (L307.4; BD Biosciences), CD86 (FUN-1; BD Biosciences), CD86 (IT2.2, BioLegend), CD25 (BC96; eBioscience), FoxP3 (236A/E7; eBioscience), CTLA-4 (BNI3; BD Biosciences), IFN-γ (B27; BD Biosciences), CD3 (UCHT1; BD Biosciences), CD4 (SK3; BD Biosciences), CD45RA (HI100; eBioscience), CD38 (HIT2; BD Biosciences) and HLA-DR (G46-6; BD Biosciences). For surface marker staining, cells were washed and re-suspended in 50 µl of FACS buffer (PBS and 2% Goat serum) containing antibodies conjugated with fluorochromes. Reactions were incubated for 30 min on ice. Cycling CTLA-4 was stained for 30 min at 37°C. For intracellular staining of FoxP3, a FoxP3 staining kit (eBioscences) was used according to the manufacturer’s instructions. Flow cytometry data were analysed by FlowJo (TreeStar, Ashland, Oregon, USA).
4
Immunophenotyping of Frozen PBMCs
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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1 mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, Biolegend), CD19 (HIB19, BioLegend), IgD (IA6-2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 min at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 min at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).
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