Blood samples were collected into ACD (citric acid, citrate, dextrose) tubes before the first pulse of MP (baseline or day 0) and the following days until the patient’s discharge from the hospital. We were unable to collect blood samples at day 3 and day 8 for the patients discharged from the hospital before this time point. Whole peripheral blood mononuclear cells (PBMCs) were isolated through a Ficoll gradient (Eurobio, Les Ullis, France) and analyzed by flow cytometry (FACS Canto II, BD Bioscience). PBMCs were surface-stained with monoclonal antibodies: PerCP-conjugated-anti-CD4, APC-H7-conjugated-anti-CD45RA, BV450-conjugated-anti CD127, PeCy7-conjugated-anti CD25 (all from BD bioscience). Cells were then fixed and permeabilized using a fix/perm buffer (eBioSciences) following the manufacturer’s instructions and then intracellularly stained with PE-conjugated-anti-FoxP3 (259D clone) and FITC-conjugated-anti-Ki67 (BD Bioscience). The FoxP3 expressing CD4+ subset phenotype was defined as previously shown [8 (link)]. Naïve Treg cells were defined as CD4+CD45RA+FoxP3low cells (nTreg) and effector Treg cells were defined as CD4+CD45RA−FoxP3high cells (eTreg), while FoxP3 expressing non-regulatory Treg CD4+ T cells were defined as CD4+ CD45RA−FoxP3low cells (non-reg FoxP3+ T cells).
Percp conjugated anti cd4
Manufactured by BD
Sourced in United States
PerCP-conjugated anti-CD4 is a fluorochrome-labeled antibody that binds to the CD4 cell surface receptor. It is used in flow cytometry applications to identify and quantify CD4-positive cells in biological samples.
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Lab products found in correlation
Ionomycin, by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
Pe cy7 conjugated anti cd25, by BD (1 mentions)
Fitc conjugated anti ki67, by BD (1 mentions)
Ficoll gradient, by Eurobio Scientific (1 mentions)
Fix perm buffer, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd11b, by BioLegend (1 mentions)
Fitc conjugated anti gr 1, by BioLegend (1 mentions)
Apc conjugated anti ifn γ, by BD (1 mentions)
Percp conjugated anti cd8α, by BD (1 mentions)
Apc conjugated anti cd11c, by BD (1 mentions)
Pe conjugated anti il 2, by BD (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Pe conjugated anti foxp3, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Fitc conjugated anti cd25, by BD (1 mentions)
Fitc conjugated anti ifn γ, by BD (1 mentions)
Ficoll hypaque density centrifugation, by Merck Group (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Golgiplug, by BD (1 mentions)
6 protocols using percp conjugated anti cd4
1
Phenotypic Analysis of Regulatory T Cells
2
Immune Cell Profiling by Flow Cytometry
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The immune cell populations were subjected to surface staining for flow cytometry analysis. The used antibodies are as follows: PE‐conjugated anti‐CD3ε (Biolegend), PerCP‐conjugated anti‐CD4 (BD Pharmingen), PerCP‐conjugated anti‐CD8α (BD Pharmingen), FITC‐conjugated anti‐NK1.1 (Biolegend), FITC‐conjugated anti‐CD11b (Biolegend), PE‐conjugated anti‐CD103 (Biolegend), APC‐conjugated anti‐CD11c (BD Pharmingen), PerCP‐conjugated anti‐F4/80 (Biolegend), or FITC‐conjugated anti‐Gr‐1 (Biolegend).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
For analyzing cytokine secretion, splenocytes were inoculated into a 12‐well plate at 4 × 106/well and incubated with 10 μg/mL CAIX protein at 37°C and 5% CO2 for 3 days, then added 500 ng/mL Ionomycin (Sigma‐Aldrich) and 50 ng/mL PMA (Sigma‐Aldrich) and 5 ng/mL Brefeldin A (BFA, eBioscience) to incubate for 5 hours. Then cells were collected and operated extracellular staining with anti‐mouse PerCP‐conjugated anti‐CD8α and intracellular staining with following anti‐mouse antibodies: APC‐conjugated anti‐IFN‐γ (BD Pharmingen), PE‐conjugated anti‐IL‐2 (BD Pharmingen), FITC‐conjugated anti‐TNF‐α (BD Pharmingen). The data acquired from BD FACSCanto II (BD Biosciences) in FACSDiva software were analyzed by FlowJo software (Tree Star Inc).
3
Quantification of Induced Regulatory T Cells
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Flow cytometric analysis was performed to quantify the induced CD4+CD25+ T cells. Cells were stained with PerCP-conjugated anti-CD4 (Clone: L200, BD Pharmingen), FITC-conjugated anti-CD25 (Clone: M-A251, BD Pharmingen) and PE-conjugated anti-FoxP3 (Clone: 206D, BD Pharmingen) respectively. Cells were stained with propidium iodide (PI, Sigma-aldrich) for nonviable cell exclusion prior to being analyzed on a FACS Calibur cytometerical (Becton Dickinson). Data processing was accomplished by CELLQuest software (BD). Cell viability measured by PI was always more than 98 % before stimulating and exceeded 95 % after stimulating.
4
Identification of TH17 Cells from PBMCs
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PBMCs were isolated with Ficoll-Hypaque density centrifugation (Sigma Aldrich, St Louis, Mo). TH17 cells were identified by means of intracellular staining of CD4+ T cells for the production of IL-17. Briefly, 1 × 106 cells from patients and an age matched healthy control subject were stimulated for 6 h with 10 ng/ml phorbol 12-myristate 13-acetate and 1 ug/ml ionomycin (Sigma-Aldrich, St Louis, Mo) in the presence of GolgiPlug (BD Biosciences, San Jose, CA). After cell-surface staining with PerCP-conjugated anti-CD4 (BD Biosciences, San Jose, CA), cells were fixed, permeabilized (Cytofix/Cytoperm, BD Biosciences, San Jose, CA), and stained with Alexa Fluor 647-conjugated anti-IL-17A (BD Biosciences, San Jose, CA). Immunoglobulin isotype control was used as a background control. CD4+ T cells were also evaluated for IFN-γ production (FITC-conjugated anti-IFN-γ; BD Biosciences, San Jose, CA). CD4+IL17+IFN-γ− cells were taken as TH17 cells (Supplementary Figure 1A ).
5
Comprehensive Blood Cell Analysis
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Venous blood sample (3 ml) was analyzed for counting total white blood cells (WBCs), platelets (Plt), and lymphocytes by hematology auto analyzer (Sysmex, KX-21N). For flow cytometry analysis, 50 μL of the samples were relocated into the round-bottom tubes to determine the CD4+, and CD8+ T cells count by flow cytometry (BD FACSCalibur, USA) according to the instructions. Cell surface antibodies were including peridinin chlorophyll protein (PerCP)-conjugated anti-CD4 and PerCP-conjugated anti-CD8 (BD Biosciences, MA, USA). A number of 10,000 leukocytes were counted for each sample and the results were analyzed using Flowjo software (Treestar, Ashland, OR, USA). Also, the MFI of CD4 and CD8 of T-lymphocytes were calculated.
6
Quantification of T Cell Subsets from Bone Marrow
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Bone marrow mononuclear cells (BMMNCs) were separated using Lymphocyte Separation Medium (HaoYang, Tianjin, China). Lymphocyte subsets were quantified by flow cytometry using the following directly conjugated mouse anti-human monoclonal antibodies: V500-conjugated anti-CD3, PerCP-conjugated anti-CD4, APC-conjugated anti-CD45RA, and PE-conjugated anti-CCR7 (BD Biosciences, San Jose, CA). After incubation, RBCs were lysed, and white blood cells (WBCs) were fixed with a lysing solution (BD Biosciences). As previously reported [17 (link), 25 (link), 35 (link)], effector T cells, naïve T cells, effector memory T cells, and central memory T cells were identified as CD45RA+CCR7−, CD45RA+CCR7+, CD45RA−CCR7−, and CD45RA−CCR7+, respectively. Multi-parameter flow cytometric analyses were performed using a BD LSRFortessa (Becton–Dickinson). Data were analyzed using BD Diva software (Becton–Dickinson).
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