Peripheral blood cells were stained with the following mAbs two times a week after transplantation: CD56 PE (B159), CD95 PE (DX2), HLA-I PE (DX17), CD3 PerCP (SP34–2), CD4 BV510 (L200), CD4 APC (L200) (BD Biosciences, San Jose, CA); CD8 APC (BW135.80), CD20 APC-Vio770 (LT20), CD25 Pacific Blue (BC96) (BioLegend), CD45RA APC-Vio770 (T6D11), CD11b Viogreen (M1/70.15.11.5), FOXP3 (236A/E7) (Invitrogen), HLA-I PE-Vio770 (REA230) (Miltenyi Biotec Inc. San Diego, CA); CD28 BV421 (CD28.2) and CD8 BV421 (RPA-T8), (BioLegend Inc. San Diego, CA). Anti-Bw6 FITC (H38, One Lambda, Canoga Park, CA) was used to assess donor chimerism. Tregs were defined as CD3+/CD4+/CD25high/FoxP3+. The same analysis was performed on liver tissue and lymph nodes on sacrifice. Data was acquired on a FACS Canto II Flow Cytometer (BD Bioscience) and analyzed using FloJo Software (TreeStar Inc. Ashland, OR).
Cd28 bv421 cd28.2
Manufactured by BioLegend
The CD28 BV421 (CD28.2) is a fluorochrome-conjugated monoclonal antibody that binds to the CD28 receptor expressed on the surface of T cells. CD28 is a costimulatory molecule that plays a crucial role in T cell activation and function.
Automatically generated - may contain errors
Lab products found in correlation
Cd3 percp sp 34 2, by BD (1 mentions)
Flojo software, by Tree Star (1 mentions)
Cd4 apc l200, by BD (1 mentions)
Cd95 pe, by BD (1 mentions)
Cd4 bv510 l200, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Benzonase endonuclease, by Merck Group (1 mentions)
Live dead aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd27 bv711, by BioLegend (1 mentions)
Ccr7 fitc g043h7, by BioLegend (1 mentions)
2 protocols using cd28 bv421 cd28.2
1
Multiparametric Flow Cytometry Analysis of Immune Cells
2
Comprehensive T Cell Phenotyping Protocol
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T cell phenotyping was conducted in Oxford for all 16 UK volunteers and 27 Senegalese volunteers for whom there were cryopreserved cells available. PBMCs were thawed and rested for 2 h at 37°C in 2.5 µl/ml Benzonase Endonuclease (E1014-25KU; Sigma-Aldrich). One to two million PBMCs for each individual were stained in 50 µl in 96-well plates. Cells were incubated for 20 min at room temperature (RT) with the Live/Dead Aqua Dead Cell Stain Kit (Invitrogen) then surface stained at RT with CD14-eF506 (61D3, 1/50; eBioscience), CD19-eF506 (HIB19, 1/50; eBioscience), CD45RA-eV605 (HI100, 1/50; eBioscience), CD27-BV711 (O323, 3/50; BioLegend), CD28-BV421 (CD28.2, 1/50; BioLegend), CD4-APC (RPA-T4, 1/50; eBioscience), CD3-AF700 (UCHT1, 1/50; eBioscience), CD8-APC-AF780 (RPA-T8, 3/50; eBioscience), CCR7-FITC (G043H7, 1/50; BioLegend), CD57-PerCP-Cy5.5 (HNK-1, 1/50; BioLegend), and KLRG1-PE (14C2A07, 3/50; BioLegend). Cells were acquired immediately using a BD LSRII. Data analysis was conducted in FlowJo version 10.1 (Treestar Inc). Gating strategy is shown in Fig. S1 .
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