Cells in BALF were stained with CD45-FITC (clone 30-F11), CD11b-APC (clone M1/70), CD11c-PE (clone N418), F4/80-PE-CY7 (clone BM8), and Ly6G-BV421 (clone 1A8, BioLegend) in the presence of an Fc blocker (CD16/CD32, BD Biosciences). A total of 2 × 106 single lung cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Cat#00-4970-03 and #00-4980-93, eBioscience) in IMDM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin for 4 h in a 37°C incubator with 5% CO2. The cells were incubated with a Zombie NIR™ Fixable Viability Kit (Cat#423105, BioLegend) at room temperature for 15 min and then stained with antibodies to extracellular antigens in the presence of Fc blocker at 4°C for 25 min. Intracellular and nuclear staining was performed with the Foxp3/Transcription buffer set (Cat# 00-5523-00, eBiosciences). Data were acquired using LSRFortessa and FACSDiva software (BD Biosciences) or Attune NxT 3 L-BRV and Attune NxT software (Thermo Fisher Scientific) and analyzed using FlowJo 10.7.2 (TreeStar, Ashland). The following antibodies were used: CD4-FITC (clone GK1.5), TCR-BV421 (clone H57-597), Foxp3-AF647 (clone MF23), IL-10-PE (JES5-16E3, BD Biosciences), IFN-γ-PE-Cy7 (clone XMG1.2), IL-4-BV605 (clone 11B11), IL-17a-BV510 (clone TC11-18H10.1, Biolegend), and IL-13-PE-eFluor 610 (eBiosciences).
Cell stimulation cocktail and protein transport inhibitor cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cell Stimulation Cocktail and Protein Transport Inhibitor Cocktail are laboratory solutions designed to facilitate the study of cellular processes. The Cell Stimulation Cocktail is used to activate cellular pathways, while the Protein Transport Inhibitor Cocktail is used to inhibit the movement of proteins within the cell. Both products are intended for research use only and their specific applications should be determined by the user.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 fitc, by BD (1 mentions)
Ly6g bv421, by BioLegend (1 mentions)
Cd11c pe, by BioLegend (1 mentions)
Ifn γ pe cy7, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Attune nxt software, by Thermo Fisher Scientific (1 mentions)
Zombie nir fixable viability kit, by BioLegend (1 mentions)
Cd16 cd32, by BD (1 mentions)
Foxp3 transcription buffer set, by Thermo Fisher Scientific (1 mentions)
Il 4 bv605, by BioLegend (1 mentions)
Il 17a bv510, by BioLegend (1 mentions)
Il 10 pe jes5 16e3, by BD (1 mentions)
F4 80 pe cy7 clone bm8, by BioLegend (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti cd4 fitc, by Thermo Fisher Scientific (1 mentions)
Anti cd25 pe, by Thermo Fisher Scientific (1 mentions)
Anti foxp3 apc, by Thermo Fisher Scientific (1 mentions)
Anti cd86 pe, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
2 protocols using cell stimulation cocktail and protein transport inhibitor cocktail
1
Multiparameter Flow Cytometry of Immune Cells
2
Immune Cell Phenotyping and Activation
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Peripheral blood mononuclear cells were treated with Cell Stimulation Cocktail and Protein Transport Inhibitor Cocktail (eBioscience, San Diego, CA, USA) and then stained with anti-CD4-FITC and anti-CD25-PE (eBioscience, San Diego, CA, USA) for surface Ags. Then the cells were permeabilized with permeabilization/fixation buffer (eBioscience, San Diego, CA, USA) and stained with anti-Foxp3-APC and anti-IL-17A-PE-Cy7 (eBioscience, San Diego, CA, USA). DCs were washed and incubated with anti-CD40-APC, anti-CD80-PE-Cy7, and anti-CD86-PE (eBioscience, San Diego, CA, USA). Cells were re-suspended for flow cytometric analysis. Flow cytometry was performed by FACS Canto II Flow Cytometer (BD Biosciences, San Jose, CA, USA) and data analysis was performed by FACS Diva software (BD Biosciences, San Jose, CA, USA).
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