The single-cell suspension was isolated from the draining lymph node (dLN) by the traditional method. Cells were cultured with 200 ng/mL of phorbol 12-myristate 13-acetate (PMA; Merck, Darmstadt, Germany) and 500 ng/mL of ionomycin (Merck) for 4 hours, which was followed by the addition of brefeldin A (BFA; 1 µg/mL; Sigma-Aldrich) 0.5 h later. After being rinsed, cells were resuspended with 50 µL of PBS, and extracellular staining was performed with 0.3 µL of CD4-fluorescein isothiocyanate (FITC; BD Bioscience, San Jose, CA, USA). Cells were then fixed and permeabilized with the intracellular fixation buffer (Fix/Perm Cell Permeabilization Kit; eBioscience, San Diego, CA, USA) for 20 min, which was followed by intracellular staining with interleukin 17 A (IL-17A) for 30 min. The relevant isotype mAb (eBioscience) was used as the fluorescence minus one (FMO) control. The positive cells were detected by the ACEA Novocyte flow cytometry (ACEA Biosciences, San Diego, CA, USA), and data were processed with the FlowJo software v. 10.4.0 (FlowJo LLC, Ashland, OR, USA)
Cd4 fluorescein isothiocyanate fitc
Manufactured by BD
Sourced in United States
CD4-fluorescein isothiocyanate (FITC) is a fluorescent dye-labeled antibody used in flow cytometry analysis. It binds to the CD4 receptor on T helper cells, allowing for the identification and enumeration of this cell population.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (2 mentions)
Facs permeabilizing solution 2, by BD (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Acea novocyte flow cytometry, by Agilent Technologies (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Fix perm cell permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BD (1 mentions)
Cd8 percp cy5, by BD (1 mentions)
Cd3 allophycocyanin apc, by BD (1 mentions)
Facscanto flow cytometry system, by BD (1 mentions)
Cd8 fitc, by BD (1 mentions)
Biotinylated recombinant protein l, by Thermo Fisher Scientific (1 mentions)
Igg1 pe, by Abcam (1 mentions)
Facscanto, by BD (1 mentions)
Cd3 pe, by BD (1 mentions)
Cd8α pe, by BD (1 mentions)
Tlr3 pe, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd14 fitc, by BD (1 mentions)
6 protocols using cd4 fluorescein isothiocyanate fitc
1
Intracellular Cytokine Staining of Th17 Cells
2
Intracellular Cytokine Staining of PBMC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMC were plated in a 96-well plate (106 cells per well). PBMC stimulation was performed in 10% FBS/RPMI media in the presence of 1 μg/ml anti-CD28 and anti-CD49d and Brefeldin A (BD Biosciences, San Diego, CA) and stimulated with HIV peptides (New England Peptide, Gardner, MA) of 15-mer overlapping by 11 amino acids representing HIV subtype E-Env (TH023; 162 peptides) and HIV subtype B-Gag (LAI; 120 peptides). PBMC supplemented with DMSO was used as a negative control. After 6 h of stimulation at 37 °C, 5% CO2 EDTA (20 mM, Sigma) was added and incubated for 15 min. Subsequently, PBMC were fixed and permeabilized using FACS lysing solution and FACS permeabilizing solution 2 (BD) according to the manufacturer’s instructions. The following antibodies were added for 60 min at room temperature in the dark: CD4-fluorescein isothiocyanate (FITC), CD3-allophycocyanin (APC), IFNγ-phycoerythrin (PE), Il-2-phycoerythrin (PE), and CD8-PerCP-Cy5.5 (all BD Biosciences). PBMC were washed and fixed with 1% paraformaldehyde. The analysis was performed using a FACSCalibur flow cytometer (BD Immunocytometry Systems). ICS data were provided to us by the trial investigators in either an ICS-positivity score (call) format and aggregate value format2 (link). ICS analytes included CD154, IFNγ, IL-4, IL-2, IL-17α, and TNFα.
3
Characterizing T Cell and Tumor Antigen Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cell samples were analyzed with a BD FACSCanto™ flow cytometry system (BD Bioscience, CA, USA), and statistical analysis was conducted in FlowJo software (FlowJo, OR, USA). The phenotype of T cells was assessed with fluorescently labeled antibodies specific for human CD3-PC5, CD4-fluorescein isothiocyanate (FITC) and CD8-FITC, which were obtained from BD Bioscience. Tumor surface antigen expression was detected with antibodies against human CD133-phycoerythrin (PE) (BioLegend, CA, USA) and GPC3-PE (Abcam, MA, USA); isotype control groups were stained with IgG1-PE (Abcam). The expression of GFP in T cells was evaluated FL1 channel to demonstrate the expression of CD133-CAR. The expression of GPC3-CAR was assessed by recombinant biotinylated protein L (Thermo Fisher Scientific) binding PE-conjugated streptavidin (PE-SA, BD Bioscience). All FACS-related cell samples were handled on ice and washed three times with 1 × PBS (Thermo Fisher Scientific) containing 1% FBS before staining the corresponding antibodies.
4
T Lymphocyte Immunophenotyping by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell suspensions were treated with ammonium chloride-potassium (ACK) buffer (150 mmol/l NH4 Cl, 10 mmol/l KHCO3 and 0.1 mmol/l disodium ethylenediamine tetraacetic acid) and then stained with the following conjugated antibodies: CD3-PE (554833, BD Biosciences), CD4-fluorescein isothiocyanate (FITC) (561834, BD Biosciences), and CD8α-PE (554857, BD Biosciences). T lymphocytes were analyzed using analyzed on a FACSCanto (BD Biosciences) running FACSDiva software (version 5.01).
5
Multiparameter Immunophenotyping of PD-1 and TLR3 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorescent dye conjugated antibodies consisted of cell surface monoclonal antibodies CD4-fluorescein isothiocyanate (FITC), CD8-peridinin-chlorophyll-protein complex (PerCP)-Cy5.5, PD-1-phycoerythrin (PE), CD25-PE and CD14-FITC (all from BD Biosciences, San Jose, CA), and intracellular monoclonal antibodies FoxP3-Alexa Flour 647 (BD Biosciences) and TLR3-PE (from eBioscience, San Diego, CA). All appropriate isotype controls were obtained from BD Biosciences. Intracellular staining of FoxP3 was performed according to the manufacturer’s staining kit (BD Biosciences) instructions. Cell surface antigens to identify the PD-1 expression on CD4+ or CD8+ T-cells were detected with CD4-FITC, CD8-PerCP-Cy5.5 and PD-1-PE. To detect intracellular TLR3 expression in monocytes, cells were stained with surface antibodies CD14-FITC, fixed, permeabilized using FACS™ permeabilizing solution 2 (BD Biosciences) and incubated with TLR3-PE.
At least 100,000 cells were acquired on FACS CantoII (Becton Dickinson, San Jose, CA) and analyzed using Cellquest software. For data analyses, an initial lymphocyte gate was set based on side scatter (SSC)/forward scatter (FSC) and additional gates introduced as required. Results were present as the percentage of positively stained cells within the gated population.
At least 100,000 cells were acquired on FACS CantoII (Becton Dickinson, San Jose, CA) and analyzed using Cellquest software. For data analyses, an initial lymphocyte gate was set based on side scatter (SSC)/forward scatter (FSC) and additional gates introduced as required. Results were present as the percentage of positively stained cells within the gated population.
6
Multiparameter Flow Cytometry for Immune Cell Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stained at 4°C for 30 minutes with the selected antibodies in phosphate buffered saline (PBS, Gibco cat# 10010-023) containing 2% fetal calf serum (Gibco, cat# 12483020) and 0.05% sodium azide (Sigma, cat# S2002). Cells were then washed and resuspended in 1% paraformaldehyde + PBS for acquisition (ThermoFisher, cat# J19943-K2). Antibodies used included CD3-phycoerythrin (PE) (BD, cat# 555333), CD4-fluorescein isothiocyanate (FITC) (BD, cat# 555346), CD8-peridinin chlorophyll protein (PerCP) (BioLegend, cat# 301030, CD56-allophycocyanin (APC) (BD, cat# 555518), CD19-FITC (BD, cat# 555412), and CD14-PerCP-Cyanine5.5 (BD, cat# 550787). Data was acquired on a FACSCalibur flow cytometer (BD) and analysed using FlowJo software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!