The eBioscience™ Cell Stimulation Cocktail (lot no.: 2046941) was purchased from Thermo Fisher Scientific Inc. (USA). FITC-conjugated anti-human CD4 (lot no.: B262223), PerCP-Cy5.5-conjugated anti-human CD3 (lot no.: B560835), Brilliant Violet 510-conjugated anti-human IFN-γ (lot no.: B323449), PE-conjugated anti-human IL-17A (lot no.: B315520), APC anti-human FOXP3 (lot no.: 1995356), PE-conjugated anti-human CD25 (lot no.: 2173867), Brilliant Violet 421-conjugated anti-human CD274 (lot no.: B320732), PE/Cyanine7-conjugated anti-human CD279 (lot no.: B318888), Fixation/Permeabilization Concentrate (lot no.: 2084746), eBioscience™ Fixation/Perm Diluent (lot no.: 2047346), Permeabilization Buffer 10× (lot no.: 2060497), and FIX&PERM (Fixation Medium A, Permeabilization Medium B) (lot no.: 17159) were purchased from Biolegend (USA).
Fitc conjugated anti human cd4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The FITC-conjugated anti-human CD4 is a monoclonal antibody that binds to the CD4 antigen on the surface of human cells. The antibody is labeled with the fluorescent dye FITC, allowing for the detection and analysis of CD4-positive cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pe conjugated anti human cd25, by Thermo Fisher Scientific (2 mentions)
Permeabilization buffer 10, by BioLegend (1 mentions)
Ebioscience cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Pe conjugated anti human il 17a, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization concentrate, by Thermo Fisher Scientific (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Pe cy5 conjugated anti humancd25, by Thermo Fisher Scientific (1 mentions)
Intracellular staining kit, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Phytohaemagglutinin pha, by Merck Group (1 mentions)
Cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Cytofix cytoperm kit, by Thermo Fisher Scientific (1 mentions)
Golgistop, by BD (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
6 protocols using fitc conjugated anti human cd4
1
Multi-color Flow Cytometry Assay
2
Phenotypic Analysis of Activated CD4+ T Cells
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In order to determine the activation of CD4+ T cells, PBMCs were incubated with FITC-conjugated anti-human CD4, APC-conjugated anti-human CD44, PE-conjugated anti-human CD25, PE-cy7-conjugated anti-human CD69 or appropriate isotypes (eBioscience, San Diego, CA) for 30 minutes at 4°C. FACScan flow cytometer (BD Biosciences, San Diego, CA) and FlowJo software (Tree Star, Inc. Ashland, USA) were used for flow cytometry analysis.
3
Quantifying Th17 and Treg Cells by Flow Cytometry
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Flow cytometry was used to measure the proportion of Th17 cells and
CD4+CD25+Treg cells in mononuclear cells in peripheral
blood. For Tregs detection, lymphocytes were stained with fluorescein
isothiocyanate (FITC)-conjugated anti-human CD4, PE-Cy5-conjugated anti-human
CD25, and PE-conjugated anti-human Foxp3 mAbs (eBioscience, San Diego, CA, USA)
and the proportion of CD25+Foxp3+ lymphocytes gated in
CD4+ lymphocytes were defined. For the detection of Th17 cells,
single-cell suspensions were stimulated with 50 ng/mL phorbol myristate acetate,
1 µg/mL ionomycin, and 2 µg/mL monensin. After 5 h, cells were stained with
FITC-conjugated anti-human CD3 and PE-conjugated anti-human CD4 mAbs, fixed,
permeabilized, and stained with PE-Cy5-conjugated anti-human IL-17A mAb
according to the intracellular staining kit (Invitrogen, Carlsbad, CA, USA)
instructions. We determined the proportion of CD4 + IL-17 + lymphocytes gated in
mononuclear cells in peripheral blood.
CD4+CD25+Treg cells in mononuclear cells in peripheral
blood. For Tregs detection, lymphocytes were stained with fluorescein
isothiocyanate (FITC)-conjugated anti-human CD4, PE-Cy5-conjugated anti-human
CD25, and PE-conjugated anti-human Foxp3 mAbs (eBioscience, San Diego, CA, USA)
and the proportion of CD25+Foxp3+ lymphocytes gated in
CD4+ lymphocytes were defined. For the detection of Th17 cells,
single-cell suspensions were stimulated with 50 ng/mL phorbol myristate acetate,
1 µg/mL ionomycin, and 2 µg/mL monensin. After 5 h, cells were stained with
FITC-conjugated anti-human CD3 and PE-conjugated anti-human CD4 mAbs, fixed,
permeabilized, and stained with PE-Cy5-conjugated anti-human IL-17A mAb
according to the intracellular staining kit (Invitrogen, Carlsbad, CA, USA)
instructions. We determined the proportion of CD4 + IL-17 + lymphocytes gated in
mononuclear cells in peripheral blood.
4
Quantification of CD4+ T cell cytokines
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Whole blood samples were collected from each subject at weeks 0 (baseline), 12, 24, 36, and 52 post LdT treatment. Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood samples using an Optipep Nycoprep Lymphoprep kit (Axis-Shield, Oslo, Norway), followed by CD4+ T cells isolation using a CD4+ T cell isolation kit (Miltenyibiotec, Germany). CD4+ T cells were maintained in RPMI 1640 medium (Hyclone, South Logan, UT, USA) supplemented with 10% foetal bovine serum (GIBCO BRL, Grand Island, NY, USA). After stimulation with 1 μg ml−1 phytohaemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO, USA), 100 U ml−1 IL-2 (eBioscience), and 1 μl ml−1 GolgiStop (BD Biosciences, San Jose, CA, USA) for 6 hours, intracellular staining for cytokines were performed using a Cytofix/Cytoperm kit (eBioscience), FITC-conjugated anti-human CD4 (eBioscience), phycoerythrin-conjugated anti-human IL-26 (R&D), allophycocyanin-conjugated anti-human RORγt (eBioscience), and isotype IgG control. The expression of each cytokine was analysed using a FACSAria cell sorter (BD Biosciences) and FlowJo single cell analysis software (Tree Star, Inc., Ashland, OR, USA).
5
Isolation and Analysis of Rat Brain Mononuclear Cells
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Mononuclear cells were isolated from rat brains as previously described. 23 Briefly, brain tissues were dissected and minced finely in RPMI 1640 and digested in 0.0625% trypsin (in Ca 2+ /Mg 2+ -free HBSS) for 20 minutes at room temperature. Single-cell preparations of the brains were suspended in 30% Percoll and banded on a 70% Percoll cushion at 900 × g at 15°C. Brain leukocytes obtained from the 30% to 70% Percoll interface were collected.
Following the preparation of single-cell suspensions, Th9 and Th22 cell proportions were evaluated. In brief, the supernatants were incubated with FITC-conjugated anti-human CD4 (eBioscience, San Diego, CA, USA) at room temperature for 20 minutes. After surface staining, intracellular staining for IL-22 (IgG1, Clone 142928, R&D, USA) and IL-9 (IgG1, Clone MH9A3, BD Pharmingen, USA) was performed with ready-to-use buffers according to the manufacturer's suggestions (BioLegend, USA). The expression of cell surface and intracellular markers was assessed using flow cytometry (LSRII, Becton-Dickinson, USA) after gating on live cells determined by scatter characteristics. Isotype controls were used to confirm appropriate compensation and antibody specificity. The data were analyzed using FlowJo software (Tree Star Inc. San Carlos, CA, USA).
Following the preparation of single-cell suspensions, Th9 and Th22 cell proportions were evaluated. In brief, the supernatants were incubated with FITC-conjugated anti-human CD4 (eBioscience, San Diego, CA, USA) at room temperature for 20 minutes. After surface staining, intracellular staining for IL-22 (IgG1, Clone 142928, R&D, USA) and IL-9 (IgG1, Clone MH9A3, BD Pharmingen, USA) was performed with ready-to-use buffers according to the manufacturer's suggestions (BioLegend, USA). The expression of cell surface and intracellular markers was assessed using flow cytometry (LSRII, Becton-Dickinson, USA) after gating on live cells determined by scatter characteristics. Isotype controls were used to confirm appropriate compensation and antibody specificity. The data were analyzed using FlowJo software (Tree Star Inc. San Carlos, CA, USA).
6
Quantifying Tregs in COVID-19 Patients
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To analyze the frequency alteration, flow cytometry was used to count the population of circulating Tregs among PBMCs in mild and severe COVID-19 infected patients as well as healthy subjects. Using monoclonal antibodies against the surface and intracellular markers. Subsequently, to cell surface staining, the harvested cells (1 × 106) were washed and incubated along with monoclonal antibodies against surface and intracellular antigens, including fluorescein isothiocyanate (FITC)-conjugated anti-human CD4, phycoerythrin (PE)-conjugated anti-human FoxP3, and allophycocyanin (APC)-conjugated anti-human CD25 (eBioscience) at 4 °C for 15 min. The FACS Calibur flow cytometer (BD Biosciences) and FlowJo software (Becton Dickinson, Mountain View, CA, USA) analyzed the population of stained cells.
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