Rosettesep nk cell enrichment kit

ELISA plates were coated with 100ul/well of pertussis, influenza A, and RSV antigen at 2 µg/mL each and incubated 2 h at 37 °C. Plates were washed three times with PBS and blocked with 5% BSA/PBS overnight at 4 °C. NK cells were isolated from Buffy coats from 3 healthy adult donors (not cord or infant blood from the study) using RosetteSep NK cell enrichment kit (StemCell Technologies) and rested overnight in the presence of IL-15. The following day, samples were diluted 1:25 and 50 µL/well diluted sample was added to ELISA plates after washing, incubating at 37 °C for 2 h. A CD107 PE-Cy5/BFA/GolgiStop (BD Biosciences, 555802) cocktail was prepared and added directly to NK cells before adding 200 µL/well NK cells at a concentration of 2.5 × 10⁵ cells/mL. Cells and plate-bound immune complexes were incubated for 5 h at 37 °C. After incubation and washing, NK cells were stained for surface markers, CD56 PE-Cy7, CD16 APC-Cy7, and CD3 Pacific Blue (BD Biosciences, 557747, 557758, 558124) in V-bottom plates. Cells were subsequently washed and incubated with PermA for 15 min followed by intracellular staining with fluorescently conjugated MIP1β PE and IFNγ FITC (BD Biosciences, 550078, 340449). NK cell activation and degranulation markers were measured by flow cytometry with the Intellicyt iQue.

To determine antibody-dependent NK cell activation, enzyme-linked immunosorbent assay (ELISA) plates (Thermo Fisher) were coated with nonbiotinylated antigen and then blocked. One hundred microliters of a 1:50 sample dilution was added to each well. NK cells were isolated from unidentified buffy coats from healthy donors using the RosetteSep NK cell enrichment kit (STEMCELL Technologies, MA, USA) and stimulated with human recombinant IL-15 (rhIL-15) (1 ng/ml; STEMCELL Technologies) at 37°C overnight. NK cells were added to the ELISA plate and incubated together with anti-CD107a (BD, clone H4A3), brefeldin A (Sigma-Aldrich, MO, USA), and monensin (BD) for 5 h at 37°C. Next, cells were surface stained for CD56 (BD, clone B159), CD16 (BD, clone 3G8), and CD3 (BD, UCHT1). After fixation and permeabilization with FIX & PERM cell permeabilization kit (Thermo Fisher), cells were stained for intracellular markers MIP-1β (BD, clone D21-1351) and IFN-γ (BD, clone B27). NK cells were defined as CD3⁻ CD16⁺ CD56⁺ and frequencies of degranulated (CD107a⁺), gamma interferon-positive (IFN-γ⁺), and MIP1β⁺ NK cells determined by flow cytometry on a LSR Fortessa (BD) (48 (link)).

ELISA plates were coated with antigen at 300 ng/well and incubated for 2 h at 37 ˚C. Plates were blocked with 5% BSA in PBS overnight at 4 ˚C. The next day, 100 μL of diluted sample were added to the plates. Plates were incubated for 2 h at 37 ˚C to form immune complexes. During the incubation, human NK cells were isolated from buffy coats using RosetteSep NK cell enrichment kit (StemCell Technologies #15065) and Ficoll separation. After the incubation, NK cells were added to the plates at 5 × 10⁴ cells/well in R10 supplemented with anti-CD107a PE-Cy5 (1:80 dilution), GolgiStop and Brefeldin A (BD Biosciences,#554724, #555802, Sigma #B7651). Plates were incubated for 5 h at 37˚C. Following the incubation, NK cells were stained for surface markers with anti-CD56 PE-Cy7, anti-CD16 APC-Cy7 and anti-CD3 Pacific Blue (1:200 dilution) (BD Biosciences, #557747, #557758, #558124). NK cells were fixed and permeabilized with Fix&Perm cell permeabilization kit (Invitrogen). Cells were incubated with anti-MIP1β PE (1:200 dilution) and anti-IFNγ FITC (1:80 dilution) (BD Biosciences, #550078, #340449) to stain for intracellular markers. Cells were acquired on a Stratedigm 1300EXi cytometer.

CEM-NKr CCR5⁺ cells (49 (link)– (link)51 (link)) (NIH AIDS reagents program) were infected with vesicular stomatitis virus glycoprotein (VSV-g)-pseudotyped HIV-1 (strain JRCSF, 0.5 IU/cell). Infection was facilitated with Polybrene (4 μg/ml) and spinoculation (800 × g for 45 min at room temperature [RT]). Cells were then incubated for 2 days at 37°C and media (RPMI supplemented with 10% fetal bovine serum [FBS], l-glutamine, and penicillin/streptomycin [Pen/Strep]) exchanged once 6 h after infection. NK cells were isolated from buffy coats from healthy donors using the RosetteSep NK cell enrichment kit (STEMCELL Technologies) and stimulated with rhIL-15 (1 ng/ml; STEMCELL Technologies) at 37°C overnight. Infected CEM-NKr CCR5⁺ cells were labeled with CellTrace far red cell proliferation kit (Thermo Fisher) before 1:50 diluted plasma sample were added together with the NK cells. After 4 h of incubation at 37°C, cells were stained with LIVE/DEAD Fixable Blue Dead Cell Stain kit (Thermo Fisher). Cells were then fixed and permeabilized with Fixation/Permeabilization Solution kit (BD) and stained for intracellular HIV-p24 (Beckman Coulter, CA, USA; clone KC57). Cell killing was calculated as frequency of viable, p24⁺ CEM-NKr CCR5⁺ cells compared to a negative control without antibodies (phosphate-buffered saline [PBS]).