The proliferation of T cells was quantified by CFSE dilution. Isolated PBMCs were labeled with 2 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; CellTrace CFSE Cell Proliferation Kit, Thermo Fisher Scientific). CFSE-labeled PBMCs (1 × 105/well) were cultured in 96 well plate with or without EVs (4 μg) derived from naive or TSG primed WJ-MSCs, in the presence of anti-CD3/CD28 microbeads (Gibco) and recombinant human IL-2 (30 U/mL, Peprotech). After 6 days of incubation, the cells were stained with human monoclonal antibodies against CD45-APC-H7 (clone 2D1), CD3-PE-Cy5 (clone HIT3α), CD4-FITC (clone RPA-T4), and CD8-BV421 (clone RPA-T8, BD Biosciences) and measured by flow cytometry. All cells were gated on 7AAD negative cells.
Cd45 apc h7 clone 2d1
Manufactured by BD
Sourced in United States
CD45-APC-H7 (clone 2D1) is a flow cytometry reagent that binds to the CD45 antigen present on the surface of leukocytes. It is labeled with the APC-H7 fluorochrome, which can be detected using flow cytometry equipment.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Anti cd3 cd28 microbeads, by Thermo Fisher Scientific (1 mentions)
Cd8 bv421 clone rpa t8, by BD (1 mentions)
Cd4 fitc clone rpa t4, by BD (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Ficoll paque solution, by GE Healthcare (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Antibody capture beads, by BD (1 mentions)
Cd4 percp cy5.5 clone okt 4, by Thermo Fisher Scientific (1 mentions)
Cd3 pacific blue clone ucht1, by BD (1 mentions)
4 protocols using cd45 apc h7 clone 2d1
1
Evaluating T Cell Proliferation by CFSE Assay
2
Characterization of Granulocytic MDSCs
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For both patients and healthy donors, granulocytes and PBMCs were freshly obtained from 3 ml of whole blood, collected in EDTA and rigorously processed within 2 hours over centrifugation on Ficoll-Paque solution (GE Healthcare Life Sciences, Cleveland, Ohio), according to manufacturer's protocol. G-MDSCs were identified in PBMCs based on the lack of HLA-DR, the dim CD33 staining, and the expression of CD66b, with CD11b and CD16 being used to differentiate between immature (CD11b− and CD16−), intermediate (CD11b+/CD16−) and mature (CD11b+CD16+) subsets (16). PBMCs (0.5 × 106 cells/tube) were stained for 15 minutes with: CD16 PerCP-Cy5.5 (clone 3G8), CD33 PE-Cy7 (clone P67.6), CD11b APC (clone D12), HLA-DR PE (clone L243), and CD45 APC-H7 (clone 2D1) all from BD Biosciences (Becton, Dickinson and Company, Franklin Lakes, New Jersey) and with CD66b FITC (clone g10f5, BioLegend, San Diego, CA).
Fluorescence acquisition was performed using a FACS Canto flow cytometer (Becton, Dickinson and Company, New Jersey): it was considered reliable when at least 0.25 × 106 CD45+ events were recorded. The analysis of flow cytometric data was performed using FlowJo software (Tree Star, Inc. Ashland, OR).
Fluorescence acquisition was performed using a FACS Canto flow cytometer (Becton, Dickinson and Company, New Jersey): it was considered reliable when at least 0.25 × 106 CD45+ events were recorded. The analysis of flow cytometric data was performed using FlowJo software (Tree Star, Inc. Ashland, OR).
3
Evaluation of Platelet Phenotype in HD and TB
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The evaluation of platelets phenotype was performed on HD and active TB using anti‐human mAbs to CD61 PerCP (Clone RUU‐PL7F12), CD62p PE (clone AC1.2), CD42b APC (clone HIP1), CD41a FITC (clone HIP8), CD42a PE (clone ALMA.16), PAC‐1 FITC (clone PAC‐1), and CD45 APC‐H7 (clone 2D1), all from BD Bioscience (San Diego, USA) and the relative isotype controls [57 (link), 58 (link)]. For the surface staining, 20 μL of platelets was used for a tube containing 20 μL of each mAb used in different combinations. After 20 min of incubation at room temperature, the samples were resuspended in PBS acquired using a FACSCANTO II (BD Biosciences). FACS plots were analyzed using FlowJo software (version 6.1.1; Tree Star, Ashland, OR, USA). The sequential gating strategy is shown in Supporting information Fig. S7 .
4
Multiparameter Immunophenotyping of T Cells
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The following anti-human antibodies were used for staining of cytobrush and whole blood samples: CD3-Pacific Blue (clone UCHT1, BD), CD4-PerCP Cy5.5 (clone OKT4, eBioscience), CD8-Horizon-V500 (clone RPA-T, BD), CD45-APC-H7 (clone 2D1, BD), CD45RO-PE (clone UCHL1, BD), HLA-DR-APC (cloneG46-6 BD-Pharmingen) (only for cytobrush samples), α4β7-FITC (clone FIB504, Biolegend), and CCR5-PEcy7 (clone 2D7/CCR5, BD). Cells were incubated with the antibody cocktail at 4°C for 30 minutes in the dark, washed with FACS wash buffer (PBS with 1% bovine serum albumin and 0.05% sodium azide), and acquired on a FACS Canto II (BD) after fixation with 2% paraformaldehyde in water. Only cervical cytobrush samples without visible red blood cells, satisfactory staining quality, and T-cell counts above 150 cells were included in the analysis. For whole blood, a cut-off of at least 10,000 lymphocyte events was applied. Fluorescent spill over compensation was conducted with antibody capture beads (BD) stained separately with the individual antibodies used in the test samples. Gating was guided for both sample types (peripheral blood and cytobrush) separately by fluorescence minus one (FMO) controls. Flow cytometry data was processed using FlowJo version 10.4 (Tree Star Inc.).
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