Anti-CD1a (HI149, eBioscience), anti-CD2 (LT2, Sorbent LTD), anti-CD3 (TB3, Sorbent LTD), anti-CD4 (OKT4, eBioscience), anti-CD5 (LT1, Sorbent LTD), anti-CD7 (LT7, Sorbent LTD), anti-CD8 (OKT8, eBiosciences), anti-CD10 (LT10, Sorbent LTD), anti-CD11c (3.9, eBiosciences), anti-CD14 (61D3, eBioscience), CD15 (HI98, eBioscience) anti-CD16 (LNK16, Sorbent LTD), anti-CD19 (LT19, Sorbent LTD), anti-CD20 (LT20, Sorbent LTD), anti-CD21 (LT21, Sorbent LTD), anti-CD22 (LT22, Sorbent LTD), anti-CD23 (LT-4F1, Sorbent LTD), anti-CD25 (BC96, eBioscience), anti-CD33 (WM53, eBioscience), anti-CD38 (LT38, Sorbent LTD), anti- CD41a (HIP8, eBioscience), anti-CD43 (eBio84-3C1, eBioscience), anti-CD45 (H130, eBioscience), anti-CD45RA (HI100, eBioscience), anti-CD45RO (UCHL1, eBioscience), anti-CD56 (LT56, Sorbent LTD), anti-CD61 (VI-PL2, eBioscience), anti-CD64 (10.1, eBioscience) anti-CD103 (B-Ly7, eBioscience), anti-HLA-DR (LN3, eBioscience), anti-human IgG kappa light chain (TB28-2, eBioscience), anti-human IgG lambda light chain (1-155-2, eBioscience), anti-human IgM (Sorbent LTD), anti-CD117 (YB5.B8, eBioscience), mouse IgG1 K isotype control (P3.6.2.8.1, eBioscience).
Anti cd45ra
Manufactured by Thermo Fisher Scientific
Anti-CD45RA is a laboratory reagent used for the identification and enumeration of CD45RA-positive cells by flow cytometry. It is a monoclonal antibody that binds to the CD45RA antigen, which is expressed on a subset of T cells and B cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti ccr7, by Thermo Fisher Scientific (3 mentions)
Anti cd8, by Thermo Fisher Scientific (2 mentions)
Anti cd4, by Thermo Fisher Scientific (2 mentions)
Anti ccr6, by Thermo Fisher Scientific (2 mentions)
Anti cd14 61d3, by Thermo Fisher Scientific (1 mentions)
Anti cd8 okt8, by Thermo Fisher Scientific (1 mentions)
Anti hla dr ln3, by Thermo Fisher Scientific (1 mentions)
Anti cd45ro uchl1, by Thermo Fisher Scientific (1 mentions)
Anti cd25 bc96, by Thermo Fisher Scientific (1 mentions)
Anti cd4 okt4, by Thermo Fisher Scientific (1 mentions)
Mem naa, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Dynabeads cd4 positive isolation kit, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse dye, by Thermo Fisher Scientific (1 mentions)
Dynabeads pan mouse igg, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva version 6, by BD (1 mentions)
Foxp3 staining buffer, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti cd45ra
1
Comprehensive Immunophenotyping Panel
2
Isolation and Proliferation of Human CD4+ T Cells
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Buffy coats were obtained from healthy donors who gave informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated using Lympholyte (Cederlane, CL5020) density gradient separation. CD4+ T cells were MACsorted from PBMCs using human anti‐CD4 magnetic beads (Miltenyi Biotech, 130–045–101), stained with carboxyfluorescein succinimidyl ester (CFSE; Life Technologies, C34554). and plated in RPMI Medium 1640 (Gibco, A10491‐01) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% Minimum Essential Medium Nonessential Amino Acid (MEM‐NAA; Gibco, 11140–035) and 0.1% 2‐mercaptoethanol. For stimulation, T cells were incubated with anti‐CD3/anti‐CD28 beads (Dynabeads Human T‐Activator, Gibco, 11131D) for 5 days. T cell proliferation was measured by flow cytometric CFSE dilution assay using LSRII Fortessa analyzer. T cell phenotype was assessed using anti‐CD45RA, anti‐CD45RO, anti‐CCR7, and anti‐CD27 antibodies (all from eBioscience).
3
CD4+ Memory T Cell Activation Assay
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PBMCs were collected by leukapheresis from anonymous volunteer donors and isolated using Ficoll Lymphocyte Separation Medium (MP Biomedicals). Purified PBMCs were cryopreserved in 10% DMS and 90% FBS in liquid nitrogen before use. CD4 + T cells were isolated from PBMCs using Dynabeads CD4 Positive Isolation Kit (Invitrogen) as per the manufacturer’s instructions. To isolate memory T cells, naive CD4 + T lymphocytes were removed by negative selection using anti-CD45RA (eBioscience) and depleted by incubation with Dynabeads Pan Mouse IgG (Invitrogen).
Isolated CD4 + memory T cells were stained with CellTrace CFSE dye (Invitrogen) at 0.5 μM for 15 min. HUVECs were treated with 50 ng mL−1 hIFN-γ for 72 h to stimulate expression of MHC class II prior to the addition of T cells. In all, 1.5 × 106 CFSE labeled CD4 + memory T cells were added to 1 × 105 endothelial cells and cultured RPMI media supplemented with 10% FBS, 2 mMl -glutamine, 100 U mL−1 penicillin, 100 ug mL−1 streptomycin for 7 days. After co-culture, T cells were collected, washed in PBS, and stained for human CD4 + (clone RRA-T4, Biolegend) in FACs-staining buffer (2% BSA, PBS) for 30 min on ice. All samples were analyzed on the Attune NxT Flow Cytometer.
Isolated CD4 + memory T cells were stained with CellTrace CFSE dye (Invitrogen) at 0.5 μM for 15 min. HUVECs were treated with 50 ng mL−1 hIFN-γ for 72 h to stimulate expression of MHC class II prior to the addition of T cells. In all, 1.5 × 106 CFSE labeled CD4 + memory T cells were added to 1 × 105 endothelial cells and cultured RPMI media supplemented with 10% FBS, 2 mM
4
Peripheral Blood Mononuclear Cell Analysis
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Blood samples were obtained from CKD or kidney transplanted patients (End Stage Renal Disease) from Tenon Hospital (Kidney Emergencies and Renal Transplantation and nephrology and hemodialysis units), Paris, France. All analyses were performed on freshly isolated peripheral blood mononuclear cells (PBMC) from 10 to 20 mL of blood by density-gradient centrifugation (Ficoll-Paque PLUS; GE Healthcare). Cell-surface staining was performed in PBS buffer containing 2% FCS and 0.01% NaN3 on ice, as previously described [12] (link), [19] (link). Cells were first stained with PBS57-loaded or empty-CD1d-tetramers (National Institutes of Health Tetramer Core Facility), then with the following directly conjugated monoclonal antibodies (eBioscience): anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD45RA, anti-CD161, anti-TCRVα 7.2 and/or anti-Foxp3. Intra-cellular analysis of Foxp3 was performed after fixation and permeabilization using Foxp3 staining buffers (eBioscience). Data were acquired on a FACSCanto II flow cytometer (BD Biosciences) with the use of FACSDiva Version 6.1.3 software (BD Biosciences) and were analyzed with the FlowJo Version 8.5.3 software (TreeStar). Lymphocyte subpopulations were analyzed within the lymphocyte gate on forward and side-scatter plots. Results were expressed in absolute numbers per mm3 of peripheral blood.
5
Isolation and Culture of Human Memory T Cells
6
Isolation and Sorting of CD8+ T Cell Subsets
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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors by density gradient centrifugation with Lymphoprep (AXIS-SHIED Rodelokka, Oslo, Norway). CD3+ and CD8+ lymphocytes were isolated by a magnetic-activated cell sorter (MACS) using CD3 Microbeads and a CD8 T cell isolation kit (MiltenyiBiotec), respectively. The cells were then labeled with anti-CD8, anti-CCR7, and anti-CD45RA (Ebiosciences) and sorted into naïve and memory CD8+ T cell subsets on a BD fluorescence activated cell sorting (FACS) Aria sorter (BD Biosciences). For extraction of TILs, tissue from lung tumor samples were minced and digested with collagenase (2.5 mg/ml collagenase I) at 37 °C for one hour. Cell suspension was then twice filtered through 100-μm and 40-μm cell strainers (BD Biosciences) to obtain single cells. TILs were isolated from single cells utilizing density gradient centrifugation with Lymphoprep and further purified using CD8 MicroBeads (MiltenyiBiotec).
7
Phenotypic Analysis of Senescent and Exhausted T Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated using coll-paque (GE Healthcare, USA) for cellular phenotypic analysis of senescent and exhausted T cells by ow cytometry. For analysis of surface markers, cells were stained in PBS containing 2% fetal bovine serum (FBS, Thermo sher, USA)
with antibodies as indicated. Then ow cytometric analysis was carried out in BD LSRFortessa X20. The gating strategy to identify T cell subsets was applied as described previously 28 . Brie y, markers related to T cell senescence and exhaustion were also monitored using PD-1 TIM3 CD28 and CD57 antibodies.
Antibodies used in this study were from BD Biosciences and eBioscience, including anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD45RA (HI100, 1:100), anti-CXCR3 (G025H7, 1:100), anti-CCR4 (L291H4, 1:100), anti-CCR6 (G034E3, 1:100), anti-CCR7 (G043H7, 1:100), anti-CD127 (A019D5, 1:100), anti-CXCR5 (RF8B2, 1:100), anti-CD28 (CD28.2, 1:100), anti-CD57 (NK-1, 1:100), anti-KLRG1 (2F1, 1:100), anti-PD-1 (EH12.2H7, 1:100), anti-TIM3 (F38-2E2, 1:100).
with antibodies as indicated. Then ow cytometric analysis was carried out in BD LSRFortessa X20. The gating strategy to identify T cell subsets was applied as described previously 28 . Brie y, markers related to T cell senescence and exhaustion were also monitored using PD-1 TIM3 CD28 and CD57 antibodies.
Antibodies used in this study were from BD Biosciences and eBioscience, including anti-CD4 (GK1.5, 1:100), anti-CD8 (53-6.7, 1:100), anti-CD25 (PC61, 1:100), anti-CD45RA (HI100, 1:100), anti-CXCR3 (G025H7, 1:100), anti-CCR4 (L291H4, 1:100), anti-CCR6 (G034E3, 1:100), anti-CCR7 (G043H7, 1:100), anti-CD127 (A019D5, 1:100), anti-CXCR5 (RF8B2, 1:100), anti-CD28 (CD28.2, 1:100), anti-CD57 (NK-1, 1:100), anti-KLRG1 (2F1, 1:100), anti-PD-1 (EH12.2H7, 1:100), anti-TIM3 (F38-2E2, 1:100).
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