Flow cytometry analysis was performed using NovoCyte 2060R (ACEA Biosciences, San Diego, CA, USA) according to previous report (17 (link)). The transduction efficiency of IM19 CAR gene into T cells was detected using a phycoerythrin (PE)-labeled anti-CAR antibody which was developed by Immunochina Pharmaceuticals for recognizing the scFv of IM19. PE-anti-CD4 and FITC-anti-CD8 antibodies (BD Biosciences, San Jose, CA, USA) were used to detect the CD4/CD8 ratio. The APC-anti-CD45RA and FITC-anti-CD62L antibodies (Biolegend, San Diego, CA, USA) were used to evaluate the ratio of memory T cells.
Anti cd45ra apc
Manufactured by BioLegend
Sourced in United States
Anti-CD45RA-APC is a monoclonal antibody conjugated with Allophycocyanin (APC) that binds to the CD45RA antigen expressed on the surface of human cells. It can be used for the identification and enumeration of CD45RA+ cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Novocyte 2060r, by Agilent Technologies (2 mentions)
Anti cd62l fitc, by BioLegend (2 mentions)
Anti cd3 bv510, by BioLegend (2 mentions)
Facscanto 2, by BD (2 mentions)
Anti cd8 fitc, by BD (1 mentions)
Anti cd4 phycoerythrin pe, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by BD (1 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Pe cy7 anti cd127, by Sony (1 mentions)
Celltrace violet ctv, by Thermo Fisher Scientific (1 mentions)
Anti ctla 4, by Thermo Fisher Scientific (1 mentions)
Anti tim3 pe, by BioLegend (1 mentions)
Anti pd l1 pe, by BioLegend (1 mentions)
Anti pd 1 apc, by BioLegend (1 mentions)
Anti cd19 pe, by BioLegend (1 mentions)
Anti cd8 fitc, by BioLegend (1 mentions)
Anti cd14 pe, by BioLegend (1 mentions)
96 well pcr plate, by Bio-Rad (1 mentions)
Live dead viability dye, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 anti cd4, by BioLegend (1 mentions)
10 protocols using anti cd45ra apc
1
Flow Cytometry Analysis of CAR-T Cells
2
Characterization of CAR-T Cell Phenotype
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Flow cytometry was used to analyze CAR expression, the CD4/CD8 ratio, and the differentiation status of the manufactured CAR-T cells. In brief, CAR-T cells (1 × 106) were suspended in 100 μL of Dulbecco’s PBS (DPBS; Thermo Fisher) and incubated with fluorescent molecule-labeled antibodies for 30 min. After washing in DPBS twice, the cells were analyzed using a flow cytometer (NOVOCYTE 2060R; ACEA Biosciences, San Diego, CA, USA). The cells were stained with allophycocyanin (APC)-anti-CD3 (BD Biosciences, San Jose, CA, USA) and fluorescein isothiocyanate (FITC)-anti-CAR that was developed by Immunochina Pharmaceuticals to specifically recognize the scFv of CAR. Phycoerythrin (PE)-anti-CD4 and FITC-anti-CD8 (BD Biosciences) were used to label CAR-T cells to determine the CD4/CD8 ratio. APC-anti-CD45RA and FITC-anti-CD62L (BioLegend, San Diego, CA, USA) were chosen to evaluate the differentiation status of CAR-T cells.
3
Multiparametric Flow Cytometry Panel for T Cell Phenotyping
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For the sorting experiments, FITC-coupled anti-CD4 and PE-coupled anti-CD25 were purchased from BD Biosciences (MountainView, CA, USA), APC anti-CD45RA was purchased from BioLegend (San Diego, CA, USA), PE-Cy5 anti-CD11c was purchased from Beckman Coulter (Fullerton, CA, USA), BV650 anti-CD3, PE-Cy7 anti-CD127 and BV510 anti-CD16 were purchased from Sony Biotechnology (San Jose, CA, USA), and Violet Blue anti-CD14 was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). For the proliferation assays, SEE was purchased from Toxin Technology Inc (Sarasota, FL, USA), CellTrace™ carboxyfluorescein succinimidyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA) and CellTrace™ Violet (CTV) was purchased from Invitrogen (Carlsbad, CA, USA). For phenotyping, PE-anti-PD-L1, PE-anti-BTLA, PE-anti-LAG3, PE-anti-TIM3, APC-anti-PD-1, and APC-anti-4-1BB were purchased from BioLegend (San Diego, CA, USA), APC anti-ICOS was purchased from Invitrogen (Carlsbad, CA, USA) and APC anti-TIGIT was purchased from Sony Biotechnology (San Jose, CA, USA). PE-anti-PD-L2 and anti-CTLA-4 were purchased from ebiosciences (San Diego, CA, USA).
4
Immunophenotyping of SARS-CoV-2-Specific T Cells
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The immunological phenotypes of PBMCs were assessed by flow cytometry using the following surface markers: BV510-anti-CD3, PerCP-Cy5.5-anti-CD4, FITC-anti-CD8, PE-anti-CD19, APC-anti-CD45RA, BV412-anti-CCR7, BV605-anti-CD56, PE-anti-CD14, APC-anti-CD16 (BioLegend). The IFN-γ-secreting T cells were detected using PE-anti-IFN-γ (Miltenyi Biotec) and BV510-anti-CD3. After stimulation with peptide for 48 h, the SARS-CoV-2 specific T cells were tested applying BV510-anti-CD3, PerCP-Cy5.5-anti-CD4, FITC-anti-CD8 and BV421-anti-CD137, and APC-anti-CD134 (BioLegend, San Diego, CA, United States). The activations of TCR-transduced-CD4+ Jurkat and TCR-transduced-CD4+ T cells were assayed using PerCP-Cy5.5-anti-CD4, APC-anti-mTCRβ, PE-anti-CD69, PE-anti-IFN-γ, and BV421-anti-CD137. In brief, cells were stained with various Abs at room temperature for 20 min. Then, the cells were washed with PBS and stained for 15 min using LIVE/DEAD viability dye (Thermo Fisher Scientific, Waltham, MA, United States). Data were acquired using the FACSCelesta cytometer and analyzed by FlowJo software. For single-cell acquisition, CD3+CD4+CD134+CD137+T cells were sorted into 96-well PCR plates (Bio-Rad) at a single-cell level using FACSAria III.
5
Phenotypic Characterization of Activated T Cells
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PBMC were purified as indicated above and activated with TransAct™ (1:100, Miltenyi-Biotec 130-111-160) and recombinant human IL2 (100 IU/ml; PeproTech). Then at days 0 and days 2-4-6 post-stimulation, the cells were stained with Fixable Viability Stain 450 (250 ng/mL; BD Biosciences), next with anti-CD3-PE-Cy5 (1/30; clone HIT3a; BD Biosciences), anti-CD4-PE-Vio770 (1/100; clone rea623; Miltenyi Biotec), anti-CD8-APC-Cy7 (1/30; clone RPA-T8; Biolegend), anti-CD25-Alexa fluor 488 (1/30; clone M-A251; Biolegend), anti-CD45RA-APC (1/30; clone HI100; Biolegend), anti-CCR7-PE (1/50; clone G043H7; Biolegend), anti-Granzyme-B-PE (1/50; clone GB12; Invitrogen), anti-PD1-Alexa 488 (1/50; clone EH12-2H7; Biolegend) and anti-KLRG1-APC (1/100; clone Rea261; Miltenyi Biotec). FACS analyses were performed on a MACSQuant Analyzer (Miltenyi Biotec) and data analyzed using FlowJo 10 software (FlowJo, LLC). All samples were gated on forward and side scatter, for singlets and for live cells.
6
Characterization of PBMC and B-LBL Subsets
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Thawed PBMCs from patients and controls were washed with PBS 1× and blocked with FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min at room temperature. Cells were then incubated for at least 30 min at 4 °C, protected from light, with the antibodies described below. For T blasts, the following antibodies were used: anti-TCRαβ-BV421 (Biolegend, San Diego, CA, USA, #306722), anti-CD4-FITC (Biolegend, #344604), anti-CD8-BV510 (Sony Biotechnology, San Jose, CA, USA #2323660), anti-CD95-BV711 (Biolegend, #305644), anti-HLA-DR-PC7 (Biolegend, #307616), anti-CD45RA-APC (Biolegend, #304112). For B-LBL: anti-CD19-FITC (BD, #555412), anti-CD95-BV711 (Biolegend, #305644), anti-HLA-DR-PC7 (Biolegend, #307616). Surface staining was detected by LSRFortessaTM X-20 SORP Cell Analyzer (BD) and analyzed with FlowJo (BD; v10.7).
7
Flow Cytometry Analysis of Immune Cells in CKD
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After the proteinuria was reached, whole blood was collected from the tail vein of CKD animals and age matched Sham animals. Red blood cells were lysed with RBC Lysis/Fixation Solution (Biolegend #422401). Fixed cells were labeled with the following antibodies; anti-RP1-BV421 (1:100, BD Biosciences #743053), anti-CD45-BV510 (1:100, Becton Dickinson BV # 740140) anti-CD45RA-APC (1:100, Biolegend #202313), anti-CD3-Alexa488 (1:50, ITK Diagnostics #201406), anti-CD4-APC/Cy7 (1:100, ITK Diagnostics #201518), anti-CD8a-PE-Cy7 (1:100, Antibodychain #25–0084–82), anti-CD11b/c-Percp/Cy5.5 (1:100, ITK Diagnostics #201820), and anti-CD68-PE (1:100, Antibodychain #MA516653). Antibody labeling was performed for 30 min at 4 °C in FACS buffer (10% FCS, 0,1% sodium azide in PBS). Subsequently the cells were washed twice using PBS. Flow cytometric analyses (≥104 events acquired in the “single cell” gate) were performed using a Becton Dickinson FACSCanto II. Gating (supplementary material, Fig. 2) was performed with fluorescence minus one and unstained controls.
8
Intracellular IL-12 Detection by Flow Cytometry
9
Multicolor Flow Cytometry for Immune Profiling
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Fluorophore-coupled mouse monoclonal antibodies (mAbs) against human CD8α eFluor 450, CD25-PerCP-efluor710/PerCP-Cy5.5, CD134 (OX-40)-APC, CD279 (PD-1)-PE-Cyanine7 and CD137 (4-1BB)-FITC were purchased from eBioscience (San Diego, CA); anti-CD3 (OKT3), anti-CD28 (clone: 15E8), anti-CD4-FITC, and anti-CD197 (CCR7)-FITC were from Miltenyi Biotec (Bergisch Gladbach, Germany); anti-CD3-APC (clone HIT3a) was from SIGMA-ALDRICH (St. Louis, MO); anti-T-bet-Alexa Fluor 647 (Clone: 4B10) and anti-CD45RA-APC were from BioLegend (San Diego, CA); anti-human IL-18/IL-1F4-Alexa Fluor 647 and recombinant human IL-18/IL-1F4 were from R&D Systems (Minneapolis, MN); rabbit anti-human IL-18, goat anti-rabbit IgG H&L-horseradish peroxidase (HRP), goat anti-rabbit IgG H&L-Alexa Fluor 488 and rabbit IgG monoclonal Isotype Control were from Abcam (Cambridge, UK); goat anti-rabbit IgG (H&L) highly cross-adsorbed-Alexa Fluor 488 was from Invitrogen (Waltham, Massachusetts, USA).
10
T-Cell Phenotyping Protocol by Flow Cytometry
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T-cell phenotype was performed by FC employing the following monoclonal antibodies (MoAbs): anti-CD3-BV510 (BioLegend, 317,332), anti-CD4-ApcH7 (BD, 560,158), anti-CD8-Percp5.5 (BioLegend, 344,710), anti-CCR7-FITC (BioLegend, 353,216), anti-CD45RA-Apc (BioLegend, 304,112), anti-PD-1-PE (BioLegend, 621,608), anti-IgM-BV421 (BioLegend, 314,516), anti-CD137-PeCy7 (BioLegend, 309,818) and 7-AAD (BD, 559,925). Cells were incubated with FC-block (BioLegend, 101,302) for 5 min and surface staining was performed at 4 °C for 15 min. At least 104 events were evaluated using a FACSCantoII flow cytometer.
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