Cytofix cytoperm kit

The cell suspension containing 2 × 10⁶ cells was harvested by centrifugation at 500 g for 3 min, and were fixed and permeabilized with 1 ml of cytofix/cytoperm (Cytofix/cytoperm kit, BD) for 10 min at 4°C. After washing twice with perm-wash (Cytofix/cytoperm kit, BD), the cells were centrifuged at 300 g for 5 min and resuspended with 150 μl of perm-wash solution. The resuspension was divided into three equal parts: the controls, the isotypes, and the experimental groups. The cells were then incubated with mouse anti-Nestin conjugated with Alexa Fluor® 647 and mouse anti-Sox2 conjugated with Alexa Fluor® 488 antibodies for 30 min in the dark, washed twice with perm-wash, and resuspended with 500 μl of PBS. The control group sample had only cells, and cells from the isotype group were incubated with the corresponding isotype antibodies instead of the specific antibodies. The data were acquired using a Beckman CytoFLEX flow cytometry system (Beckman Coulter, USA) running the CytExpert software.

Paired samples of mononuclear cells from PB and IVB were thawed and 1x10⁶ mononuclear cells were activated for 4h at 37°C with either phorbol myristate acetate (PMA) and Ionomycin (25 ng/ml and 1µg/ml respectively (Sigma-Aldrich)) or lipopolysaccharide (LPS) (100 ng/ml (Sigma-Aldrich)). Golgistop™ (1:500 dilution, BD Biosciences, Franklin Lakes, NJ) and Brefeldin-A (1:1500 dilution, Sigma-Aldrich) were used to prevent cytokine release. Following activation, the mononuclear cells were stained for extracellular markers as described previously. After the extracellular staining, the cells were washed in FACS-buffer and resuspended in 100 µl fixation/permeabilization buffer (Cytofix/Cytoperm™ kit (BD)) and left overnight at 4°C. The next day, cells were centrifuged and washed twice with Permeabilization/Wash solution (Cytofix/Cytoperm™ kit (BD)) diluted 1:10 in dH₂O and stained with fluorochrome conjugated antibodies for intracellular markers (Supplementary Table I) for 30 minutes at 4°C. This was followed by a final wash before being resuspended in 200 µl Permeabilization/Wash solution and analysed with multicolour flow cytometry.

First, 100,000 cells were inoculated in 24-wells plates with a volume of 500 µL. The cells were incubated at 37°C for 24 h under different conditions of hypoxia (21%, 3.5%, 1% and 0.1%). BrdU at the final concentration of 30 µg/L was added before the termination of culture. Then, 2 h later, the cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen). For the pulse-chase, 100,000 cells were inoculated in 24-wells plates with a volume of 500 µL. The cells were incubated at 37°C for 24 h under 21% O₂. BrdU at a final concentration of 30 µg/L was added, and, 2 h later, the cells were rinsed and the solution was replaced. The cells were incubated at 37°C for 24, 34, 48, 58, or 72 h under different conditions of hypoxia (21%, 3.5%, 1% and 0.1%). The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer’s protocol and stained with the following antibodies: FITC-anti-BrdU antibody (BD), and APC-anti PLZF antibody (R&D). Cell death was measured using Hoechst 33258 to stain dead cells. Analyses were performed on LSR II and FACSCalibur flow cytometers (Becton Dickinson).

Treg cells were identified as anti-CD4-positive and anti-CD25-positive. Where indicated, additional markers were evaluated using anti-Human Ki-67 PerCP-eFluor® 710, human Neuropilin-1 PerCP MAb, anti-Human CD152 (CTLA-4) PE, HU FOXP3 APC, and Gata3 PE. Before intracellular staining, Cytofix/Cytoperm Kit and Pharmingen Leukocyte Activation Cocktail with BD GolgiPlug were used for cell fixation and permeation.
PE Annexin V Apoptosis Detection Kit I was used to identify the apoptosis of Treg cells. For intracellular cytokine production, PBMCs were stimulated with Leukocyte Activation Cocktail with BD GolgiPlug for 3 hours before staining. Cytofix/Cytoperm Kit and Pharmingen Leukocyte Activation Cocktail with BD GolgiPlug were used for intracellular cytokine production. Intracellular staining was performed using APC Rat anti-Human IL-4, PE Rat anti-Human IL-5, APC Rat anti-Human IL-13, PE Mouse anti-Human IFN-γ, APC Rat anti-Human IL-10, and PE Mouse anti-Human TGF-β1.
Samples were detected with a FACS flow cytometer, and acquired data were analyzed with FlowJo software.

10⁶ cells from the spleen, axillary lymph nodes, and lungs were plated in 24 well culture plates, stimulated with ConA (1 μg/mL), CMX (10 μg/mL) or without stimulus (medium) and incubated for 4 h in a 5% CO₂ chamber at 37°C. Then, the plates were incubated for another 6 h with 3 mM monensin (eBioscience). After that, the cells were labeled with FITC-anti-CD4 (GK1.5 clone) for 30 min. Subsequently, the labeled cells were fixed with Perm Fix (BD Cytofix/Cytoperm Kit) for 20 min, and their membranes were permeabilized with Perm wash (BD Cytofix/Cytoperm Kit) for 20 min. After this step, the cells were labeled with the intracellular antibodies: APC-anti-IFN-γ (clone XMG1.2) incubated for 30 min and fixed with Perm Fix. After labeling the cells, 50,000 events were acquired in the Biosciences BD FACSVerseTM flow cytometer and the data were analyzed with FlowJo 9.0 software.

Samples were resuspended in FACS buffer (PBS, 0.5% w/v BSA, 2 mM EDTA) containing 2 mg/ml human IgG before being incubated on ice in the dark for 30 min in the presence of fluorescently-labelled antibodies or isotype controls (all BD Biosciences, Oxford, UK). Intracellular staining for viral nucleoprotein (NP)-1, was performed using BD Cytofix/Cytoperm kit according to manufacturer’s instructions, and AlexaFluor 488 (AF488)-conjugated anti-NP1 antibody (HB-65, a kind gift of 3VBiosciences). Intracellular staining for RSV F protein was performed using BD Cytofix/Cytoperm kit according to manufacturer’s instructions, RSV-F protein antibody (Meridian Life Science Inc). After incubation on ice for 30 min, MDM were washed and incubated with AlexaFluor 488 anti-mouse antibody (Invitrogen, Paisley, UK). Flow cytometric analysis was performed on a FACSAria using FACSDiva software v5.0.3 (all BD).