Peripheral blood mononuclear cells (PBMCs) were isolated from 10 mL blood samples collected in EDTA–anti-coagulated tubes by standard Ficoll–Hypaque density centrifugation (Cederlane, Canada). Furthermore, CD4+/CD25+, the surface markers of PBMCs were stained by incubation with PE-CY5 conjugated anti-CD4+ (Ebioscience, Germany) and FITC conjugated anti-CD25+ (BD, Ebioscience, USA) at room temperature for 30 minutes. The FITC-mouse IgG1, PE-mouse IgG1 and PE-CY5-mouse IgG1 (BD Ebioscience. USA) were used as isotype controls. To analyze CD4, CD25 and FoxP3 expression, cells were stained with PE-CY5 conjugated anti-CD4 (Ebioscience) and FITC conjugated anti-CD25 (BD Ebioscience); followed by intracellular staining with anti-FoxP3 staining set (BD Ebioscience, USA). After staining, the cells were analyzed by flow cytometer using the Cell Quest software (Coulter, USA). The CD4+ CD25+ lymphocyte population was gated for FoxP3 expression analysis. The percentage of CD4+ CD25+ FoxP3 T cells to total CD4+ T cells, and proportion of FoxP3 cells in CD4+ CD25+ T cells were computed for different groups.
Fitc conjugated anti cd25
Manufactured by BD
Sourced in United States
FITC-conjugated anti-CD25 is a fluorescently labeled monoclonal antibody that binds to the CD25 receptor, also known as the interleukin-2 receptor alpha chain. This product is designed for use in flow cytometry applications to identify and quantify cells expressing the CD25 marker.
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Lab products found in correlation
Facscalibur, by BD (2 mentions)
Pe conjugated anti cd45ro, by BioLegend (2 mentions)
Pe conjugated anti cd69, by BD (2 mentions)
Pe conjugated anti foxp3, by BD (2 mentions)
Cell quest software, by Beckman Coulter (1 mentions)
Pe cy5 conjugated anti cd4, by Thermo Fisher Scientific (1 mentions)
Pe cy5 mouse igg1, by BD (1 mentions)
Fitc conjugated anti cd3, by BD (1 mentions)
Fcs express v3, by De Novo Software (1 mentions)
Pe conjugated anti foxp3 mab, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Dihydroethidium, by Merck Group (1 mentions)
Lymphoprep, by Axis-Shield (1 mentions)
Monochlorobimane, by Merck Group (1 mentions)
D cysteine, by Merck Group (1 mentions)
Facsymphony a5 cell analyzer, by BD (1 mentions)
12 protocols using fitc conjugated anti cd25
1
Isolation and Characterization of Regulatory T Cells
2
Cell Surface Molecule Expression Analysis
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To examine the expression level of molecules on the cell surface, cells harvested after the MLR were washed with PBS containing 2% FBS and then stained with the following antibodies (Abs): phycoerythrin-cychrome 5 (PC5)-conjugated anti-CD8 (Beckman Coulter, Inc., Brea, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-CD4, FITC-conjugated anti-CD25 (Becton Dickinson, Franklin Lakes, NJ, USA), PE-conjugated anti-CD45RA, or PE-conjugated anti-CD45RO (BioLegend, San Diego, CA, USA) at room temperature in the dark for 30 min. Cells were then washed with PBS containing 2% FBS and resuspended in 0.3 ml of PBS containing 2% FBS for analysis by flow cytometry (FCM) using FACS CaliburTM (Becton Dickinson) as previously described [14 ].
3
Cell Surface and Intracellular Protein Expression
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To examine the expression level of molecules on the cell surface, cells harvested after the MLRs were washed with PBS containing 2% FBS and then stained with the following Abs: PC5-conjugated anti-CD8 and fluorescein isothiocyanate- (FITC-) conjugated anti-CD3, FITC-conjugated anti-CD25 (Becton Dickinson), phycoerythrin- (PE-) conjugated anti-CD45RA, or PE-conjugated anti-CD45RO (BioLegend, San Diego, CA, USA) at room temperature in the dark for 30 min. Cells were then washed with PBS containing 2% FBS and resuspended in 0.3 mL of PBS containing 2% FBS for FCM analysis. To examine the expression level of intracellular granzyme B, cells were harvested after the MLRs and surfaces were stained with PC5-conjugated anti-CD8 Ab as described above. Surface stained cells were washed with PBS containing 2% FBS and then fixed with 3.7% formaldehyde for 15 min. Fixed cells were washed with PBS containing 2% FBS. Fixed cells were permeabilized with 0.1% Triton 100 and stained with R-phycoerythrin- (RPE-) conjugated anti-granzyme B Ab (AbD Serotec, Oxford, UK) at room temperature in the dark for 30 min. Cells were then washed and resuspended as described above. The percentage of cells positive for each parameter was analyzed using FCM. Four independent experiments were performed.
4
Quantification of T-Cell Subsets
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Peripheral blood samples (150 µL) were incubated with APC-conjugated anti-CD3 (3.5 µL), PerCP-conjugated anti-CD4 (3.5 µL) or PerCP-conjugated anti-CD8 (3.5 µL) and FITC-conjugated anti-CD25 (3.5 µL) monoclonal antibodies (mAbs) (BD Biosciences) at 4°C for 20 min. RBC lysis buffer (1 mL) (Boster Biosciences) was then added, and the samples were incubated for another 10 min in the dark. The tubes were centrifuged at 200×g for 5 min. The supernatants were discarded, and the cells were washed in PBS. The cells were resuspended in Foxp3 fixation/permeabilization concentrate and diluent solutions (500 µL) (eBioscience) and incubated at room temperature in the dark for 30 min. The tubes were centrifuged at 200×g for 5 min, the supernatants were discarded, and the cells were then washed twice in PBS. The cells were incubated with PE-conjugated anti-FoxP3 mAb (7 µL) (eBioscience) at 4°C for 30 min. Isotype-matched mAbs were used as negative controls during each staining stage. After washing with PBS, the stained cells were analyzed using flow cytometry with a FACS Calibur (BD Biosciences) and analyzed with FCS Express V3 software (De Novo Software).
5
Apoptosis Induction and Detection Reagents
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The following chemicals were obtained from Sigma Aldrich (USA): Glutathione (GSH), L-cysteine, D-cysteine, N-acetylcysteine (NAC), L-Buthionine-(S,R)-sulfoximine (BSO), monochlorobimane (MCB) and dihydroethidium (DHE). Monoclonal antibody (mAb) against CD3 (clone OKT3) was purified from hybridoma (ATCC) culture supernatants. Lymphoprep was from Axis-Shield PoCAS (Norway) while RPMI 1640 and FCS were from Gibco (UK). FITC-conjugated anti-CD25 and PE-conjugated anti-CD69 were acquired from BD Pharmingen (UK). The 5-bromo-2'-deoxyuridine (BrdU) labelling kit was obtained from Roche (Switzerland). Rabbit antibodies to caspase-3, mouse antibodies to β–actin and goat antibodies to caspase-8 were all from Santa Cruz Biotechnology (USA). All secondary HRP-conjugated antibodies were purchased from Dako (UK). Benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK), benzyloxycarbonyl-tyrosine-valine-alanine-aspartic acid- fluoromethylketone (z-YVAD-FMK), benzyloxycarbonyl-valine-arginine-proline-DL-arginine-fluoromethylketone (z-VRPR-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were purchased from Bachem (Switzerland). Benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) and biotinylated-phenylalanine-alanine-fluoromethylketone (b-FA-FMK) were from MP Biomedicals (USA).
6
Multi-Marker Flow Cytometry Analysis
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Peripheral blood was collected and treated with Gey’s solution to remove red blood cells. Then, the remaining cells were resuspended in phosphate-buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS). These cells were stained with a combination of fluorescence-conjugated antibodies. Antibodies, including APC-conjugated anti-B220 (553092) and anti-CD4 (553051), PE-Cy7-conjugated anti-Mac-1 (561098), PE-conjugated anti-Gr-1 (561084), BUV805-conjugated anti-CD8 (612898), and FITC-conjugated anti-CD25 (553072), were purchased from BD Biosciences. Alexa Fluor 700-conjugated anti-CD3 (100216) was purchased from Biolegend. The samples were analyzed using the FACSymphony™ A5 cell analyzer (BD Biosciences, Franklin Lake, NJ, USA), and the data were analyzed using FlowJo v10.8 software.
7
Induction of Regulatory T Cells from Naive CD4+ T Cells in SLE
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Naive CD4+ T cells were isolated from SLE and matched HC subjects by using Human Naive CD4+ T cell Enrichment Kit (STEMCELL, Cat# 19555). The purity of naive CD4+ T cells as defined by the proportion of CD4+CD45RA+CD62L+ cells was above 99%. Cells were cultured for 72 h in the presence of anti-CD3/CD28 and TGF-β (5 ng/ml, Peprotech, Cat# 100-21) with IL-2 (50 IU/ml) or anti-IL-2 (100 or 1000 ng/ml). Cells were stained with FITC-conjugated anti-CD25 (Clone: M-A251, Cat# 555431, RRID : AB_395825) and AF-647-conjugated anti-FoxP3 (Clone: 259D/C7, Cat# 560045, RRID : AB_1645411 both from BD Biosciences). Frequency of CD4+CD25+FOXP3+ cells was determined by flow cytometry. In other experiments, CD4+ T cells isolated from matched SLE and HC subjects were cultured for 3 days in the presence of anti-CD3/CD28 and TGF-β (20 ng/ml) with or without IL-2 (100 IU/ml) or anti-IL-2 (100 ng/ml).
8
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.
9
Multiparametric Analysis of T Cell Subsets
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Samples were stained with the following antibodies: FITC-, eFluor450-, and APC-conjugated anti-CD8a (Clone 53-6.7, eBioscience, San Diego, CA, USA), FITC conjugated anti-CD4 (Clone GK1.5, eBioscience), APC/Cy7-conjugated anti-CD62L (Clone MEL-14, BioLegend, San Diego, CA, USA), APC-conjugated anti-CD44 (Clone IM7, eBioscience), Alexa Fluor 647- and eFluor 450-conjugated anti-CD3 (Clone 17A2, eBioscience), FITC- and PE-conjugated anti-Kb (Clone AF6-88.5.5.3, eBioscience), FITC-conjugated anti-CD25 (Clone 3C7, BD Pharmingen, USA), and FITC-conjugated Mouse Vβ TCR Screening Panel (BD Pharmingen).
10
Multi-parameter Flow Cytometry Analysis of T-cell Phenotype and Activation
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Fluorescein (FITC)-conjugated anti-CD25 (catalog number 555431), phycoerythrin (PE)-conjugated anti-CD132 (catalog number 555900), allophycocyanin (APC), peridinin-chlorophyll proteins (PerCP)- and Pacific Blue (PB)-conjugated anti-CD4 (catalog numbers 555349, 347324 and 558116, respectively), PerCP- or FITC-conjugated anti-CD8 (catalog numbers 347314 and 555634, respectively), Alexa Fluor 647-conjugated anti-CD127 (catalog number 558598), FITC-conjugated anti-CD45RA (catalog number 555488), PE-conjugated anti-phosphorylated STAT5 (612567), PE Cy7-conjugated anti-PD-1 (catalog number 561272), Alexa Fluor 488-conjugated anti-IFN-γ (catalog number 557718) and Fixable Viability 510 (564406) were purchased from BD Biosciences. PE-conjugated anti-Bcl-2 (MHBCL04) was purchased from Thermo Fisher Scientific. Cell samples were acquired on a FACS Aria II flow cytometer (BD, USA) and analyzed with FlowJo software (Tree Star, San Carlos, CA, USA). Lymphocytes were gated based on their forward scattering and side scattering parameters, followed by the use of forward scatter area vs. forward scatter height dot-plot for doublet discrimination. The subsequent analyses were performed on viable cells (FV510—) (S1A Fig )
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