Fix perm cell permeabilization kit

Human NK cells were enriched from fresh peripheral blood of healthy human volunteer donors’ buffy coats, obtained by Clínica Sanatorio Alemán blood bank at Concepción, Chile.
Briefly, a negative selection kit (Miltenyi) was used to isolate NK cells. 2 µg /ml of SARS-CoV-2 spike recombinant proteins were coated on ELISA High Bind Microplate (Corning) at 4°C overnight, and plates were blocked with 5% bovine serum albumin (BSA) prior to addition of the serum dilutions (1/100) in PBS for 2 hours at 37°C. Unbound antibodies were removed by washing wells 3X with PBS prior to the addition of NK cells. The NK cells were added at 5 x 10⁵ cells/well in the presence of brefeldin A (BioLegend), monensin (BioLegend), and anti-CD107a phycoerythrin (PE) (BioLegend) and incubated for 5 hours at 37°C. NK cells were surface stained with CD56 Alexa Fluor 647 (BioLegend), followed by intracellular staining with IFNγ Alexa Fluor 488 (BioLegend) and MIP1β Brilliant Violet 421 (BioLegend) using the Fix & Perm cell permeabilization kit (Invitrogen) used to detect the production of cytokine/chemokine. Cells were analyzed on a BD LSRFortessa X-20 flow cytometer and data was analyzed using FlowJo software (37 (link)).

Human NK cells were isolated from buffy coats using RosetteSep NK cell enrichment kit (StemCell Technologies) and Ficoll separation. The isolated NK cells were rested overnight at 1.5 × 10⁶ cells/mL in IL-15 at 37°C. ELISA plates were coated with antigen at 300 ng/well and incubated for 2 hours at 37°C. Plates were blocked with 5% BSA in PBS overnight at 4°C. The next day, 100 μL of antibodies, at a concentration of 5 μg/mL, were added to the plates. Plates were incubated for 2 hours at 37°C to form immune complexes. After the incubation, NK cells were added to the plates at 5 × 10⁴ cells/well in R10 supplemented with anti-CD107a PE-Cy5, Brefeldin A (MilliporeSigma, B7651-5MG), and GolgiStop (BD Biosciences, 555802). Plates were incubated for 5 hours at 37°C. Following the incubation, NK cells were stained for the surface markers with anti-CD56 PE-Cy7, anti-CD16 APC-Cy7, and anti-CD3 Pacific Blue (BD Biosciences, 557747, 557758, 558124). NK cells were fixed and permeabilized with Fix&Perm cell permeabilization kit (Invitrogen, Thermo Fisher Scientific). Cells were incubated with anti–MIP-1β PE and anti–IFN-γ FITC (BD Biosciences, 550078, 340449) to stain for intracellular markers. Cells were acquired on an Intellicyt iQue.

To study the proliferation of HSPCs, mice were injected with 10 mg/ml 5-brom-2′-deoxyuridin (BrdU, Sigma Aldrich, # B9285-250MG) as early as 1 hour before harvest. BM cells were obtained and prepared for flow cytometric analysis as described above. To analyze the proportion of BrdU in the cell populations, and therefore proliferating cells, further staining was performed after the primary antibody staining. Since BrdU is incorporated into DNA and is therefore an intracellular marker, cells were fixed and permeabilized using a Fix & PERM^™ cell permeabilization kit (Invitrogen, #GAS004). The cells were then treated with deoxyribonuclease (DNase) to release the BrdU incorporated into the DNA. Cells were then stained with PE anti-BrdU antibody (Biolegend, San Diego, USA, #364116). Proliferating cells were identified as BrdU positive (BrdU⁺).

Cells were fixed and permeabilized with FIX & PERM Cell Permeabilization Kit (Invitrogen) and subsequently stained with CD45-KO (Beckman Coulter) and p-STAT1-PE or p-STAT3-PE (eBioscience). Samples were measured on the Beckman Coulter CytoFLEX. During analysis, cells were gated on being CD45⁺ to eliminate debris. Gating on monocyte and lymphocyte populations was done in the forward versus side scatter plot.