Cytofix cytoperm fixation permeabilization solution kit

Spleen cell suspensions from each mouse were obtained by dissociation of tissue and red blood cells were lysed with 0.85% ammonium chloride solution. To analyze T cells subsets, one million splenocytes were stained for 15 minutes with the following antibodies: anti-CD3-BV605, anti-CD4-PerCP-Cy5.5, anti-CD44-PE-Cy7 and anti-CD62L-APC (BD Biosciences). After incubation, the cells were washed with PBS and fixed with 1% formaldehyde in PBS. To analyze Treg cells, 1X10⁶ splenocytes were stained with anti-CD3-BV421, anti-CD4-APC and anti-CD25-APC-Cy7. To detect intracellular FoxP3 expression, cells were permeabilized (Cytofix/Cytoperm TM Fixation/Permeabilization Solution Kit, BD Biosciences) and anti-FoxP3-Alexa488 was added and incubated for 30 minutes. Data were acquired in BD LRS Fortessa flow cytometer (Becton-Dickinson, San Jose, CA) and analyzed by FlowJo software (FlowJo, Tree Star, Ashland, OR).

For surface staining, cells were stained with anti-CD3 PerCP-Cy5.5, anti-CD4 APC, anti-CD25 APC-Cy7, anti-CD69 Pe-Cy7, anti-PD-1 BV421, and anti-CD38 Alexa Fluor 488 (all from BD Biosciences, San Diego, CA, USA). Cells were then incubated for 15 minutes under room temperature and were fixed with 1% formaldehyde for 15 minutes. For intracellular staining, cells were permeabilized (Cytofix/Cytoperm TM Fixation/Permeabilization Solution Kit, BD Biosciences) for 40 minutes and stained with anti-FoxP3 Alexa Fluor 488, anti-CTLA-4 BV605, and anti-IL-10 APC. All the samples were analyzed using an LSRFortessa flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analyzed by FlowJo software (FlowJo, Tree Star, Ashland, OR, USA).

The antiviral activity of the LQM334 compound was confirmed by intracellular flow cytometry staining [119 (link)]. Briefly, after CHIKV adsorption in Vero E6 cells for 2 h, the medium was removed, the cell monolayer was washed with PBS. The LQM334 compound was added at 20 or 40 μM concentrations and the cells were maintained at 37 °C/5% CO₂ atmosphere for 48h. The cells were detached by using a trypsin/EDTA solution and submitted to fixation and permeabilization using the BD Cytofix/ Cytoperm^TM Fixation/Permeabilization solution kit (BD Biosciences^®, San Jose, CA, USA) according to the manufacturer’s recommendations. Subsequently, the cells were incubated with an anti-CHIKV monoclonal antibody (1:50; A54Q clone; Invitrogen, Carlsbad, CA, USA) for 1h at 4 °C. The cells were washed with the BD Perm Wash solution (BD Biosciences^®, San Jose, CA, USA) and then incubated with the goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody conjugated with Alexa Fluor 488 (1:200; Invitrogen) for 1h at 4 °C. A total of 20,000 events were acquired in the BD FACS Canto^TM II flow cytometer (BD Biosciences^®, San Jose, CA, USA) and the results were analyzed by using FlowJo^TM v. 10 software.

Cytokine production was analyzed by intracellular staining (ICS) assay. PBMCs were incubated with or without the relevant peptides (20 μg/ml) plus anti-CD28 mAb (4 μg/ml) (BD Biosciences) and the Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; eBioscience), or with the Cell Stimulation Cocktail (phorbol 12-myristate 13-acetate [PMA], ionomycin, brefeldin A, and monensin; eBioscience) as a positive control, for 18 h at 37°C [16 (link),22 (link)]. Cells were washed and then stained with APC-labeled—HLA-A*0201 dextramers complexed to corresponding peptides, PeCy7-labeled mAb to CD8 (BioLegend), and the dump channel reagents. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) at 4°C for 20 min, rewashed with the BD Perm/Wash buffer (BD Biosciences), and stained with different combinations of AlexaFluor700-labeled IL-17A (BioLegend) and FITC-labeled anti-IFN-γ (BioLegend) for 20 min at 4°C. Cells were washed, acquired with the LSRFortessa cytometer, and analyzed with FlowJo software. IL-17, IFN-γ or IL-17/IFN-γ producing cells were analyzed in CD8⁺dextramer⁺ cells after exclusion of B cells, monocytes, NKT cells, NK cells, and CD4⁺ T cells (dump channel).