For analysis of NK-cell related responses, an ELISA-based assay was used. Therefore, 96-well ELISA plates (Thermo Fisher) were coated with SARS-CoV-2 S at 37°C for 2h. Plates were then washed and blocked with 5% BSA in PBS overnight at 4°C. NK cells were isolated from buffy coats from healthy donors (MGH blood donor center) using the RosetteSep isolation kit (Stem Cell Technologies) and NK cells were rested overnight supplemented with IL-15 (Stemcell). Serum samples were diluted 1:50 and incubated at 37°C for 2h on the ELISA plates. A staining cocktail of anti-CD107a-PE-Cy5 stain (BD), brefeldin A (Sigma), and GolgiStop (BD) was added to the NK cells and 5×104 NK cells per well were added and incubated for 5h at 37°C. NK cells were fixed and permeabilized using Perm A and B (Thermo Fisher) and surface markers were stained for with anti-CD16 APC-Cy7 (BD), anti-CD56 PE-Cy7 (BD) and anti-CD3 AlexaFluor 700 antibodies (BD). Intracellular staining included anti-IFNγ APC (BD) and anti-MIP-1β PE (BD). Acquisition occurred by flow cytometry iQue (Intellicyt), equipped with a robot arm (PAA). NK cells were defined as CD3-, CD16+ and CD56+. The ADNKA assay was performed in duplicate across two blood donors.
Il 15
Manufactured by STEMCELL
Sourced in Canada, Germany
IL-15 is a recombinant human cytokine that stimulates the proliferation and activation of natural killer (NK) cells and T cells. It is commonly used in cell culture research applications.
Automatically generated - may contain errors
Lab products found in correlation
Brefeldin a, by Merck Group (3 mentions)
Golgistop, by BD (3 mentions)
Anti ifn γ apc, by BD (2 mentions)
Anti mip1β pe, by BD (2 mentions)
Anti cd16 apc cy7, by BD (2 mentions)
Anti cd107a pe cy5 stain, by BD (2 mentions)
Perm a and b, by Thermo Fisher Scientific (2 mentions)
Anti cd3 alexafluor 700 antibodies, by BD (2 mentions)
96 well elisa plate, by Thermo Fisher Scientific (2 mentions)
Rosettesep isolation kit, by STEMCELL (2 mentions)
Anti cd56 pe cy7, by BD (2 mentions)
α minimal essential medium, by Welgene (2 mentions)
Il 21, by STEMCELL (2 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (2 mentions)
Interleukin 2 (il 2), by STEMCELL (2 mentions)
Hydrocortisone hc, by STEMCELL (1 mentions)
Cd56 magnetic beads, by Miltenyi Biotec (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Interleukin 7 (il 7), by STEMCELL (1 mentions)
Immunocult human cd3 cd28 cd2 t cell activator, by STEMCELL (1 mentions)
8 protocols using il 15
1
SARS-CoV-2 S Protein-Induced NK Cell Responses
2
Expansion and Activation of Human NK Cells
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Human NK cells were isolated from the peripheral blood of healthy donors using RosetteSep (Stem Cell Technologies, Vancouver, BC, Canada)—which depletes cluster of differentiation 3+ T cells and red blood cells—followed by CD56 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were cultured in α Minimal Essential Medium (Welgene, Gyeongsan, Korea) with IL-15 (30 ng/ml), IL-21 (30 ng/ml), and 10−6 M of hydrocortisone (HC; Stem Cell Technologies, Canada). To investigate the effect of PGE2 on NK cell toxicity, the cells were cultured for 48 h with either control medium or thyroid cancer cell culture supernatant at 1/4 dilution.
3
Activating and Expanding Primary Human T Cells
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This research
was performed in accordance with the Declaration of Helsinki. Human
peripheral blood was obtained from healthy adults after obtaining
informed consent (Technical University of Denmark-Rigshospitalet National
Hospital approval BC-40). T cells were isolated from the PBMC population
by immune-magnetic negative selection using the EasySep Human T Cell
Isolation Kit (STEMCELL Technologies). After isolation, T cells were
activated in 25 μL ImmunoCult Human CD3/CD28/CD2 T-Cell Activator
(STEMCELL Technologies) per 1 mL ImmunoCult-XF T Cell Expansion Medium
(STEMCELL Technologies) containing 12.5 ng mL–1 of
Human Recombinant IL-2, 5 ng mL–1 of IL-7, and 5
ng mL–1 of IL-15 (STEMCELL Technologies) and seeded
at 1.0 × 106 cells mL–1 until transfection
48 h later. The cells were maintained at 37 °C in 5% CO2 incubators.
was performed in accordance with the Declaration of Helsinki. Human
peripheral blood was obtained from healthy adults after obtaining
informed consent (Technical University of Denmark-Rigshospitalet National
Hospital approval BC-40). T cells were isolated from the PBMC population
by immune-magnetic negative selection using the EasySep Human T Cell
Isolation Kit (STEMCELL Technologies). After isolation, T cells were
activated in 25 μL ImmunoCult Human CD3/CD28/CD2 T-Cell Activator
(STEMCELL Technologies) per 1 mL ImmunoCult-XF T Cell Expansion Medium
(STEMCELL Technologies) containing 12.5 ng mL–1 of
Human Recombinant IL-2, 5 ng mL–1 of IL-7, and 5
ng mL–1 of IL-15 (STEMCELL Technologies) and seeded
at 1.0 × 106 cells mL–1 until transfection
48 h later. The cells were maintained at 37 °C in 5% CO2 incubators.
4
Expansion and Purification of Human NK Cells
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Human PB NK cells were cultured on 6-well cell culture plates in Immunocult-XF T cell expansion medium (StemCell Technologies, Vancouver, BC, Canada) supplemented with 1× penicillin/streptomycin (VWR, Radnor, PA), 500 IU mL−1 IL-2 (StemCell Technologies), 0.2 μL mL−1 CD2/CD3/CD28 T cell activator (StemCell Technologies), and 10 ng mL−1 IL-15 (StemCell Technologies). Culture medium was replaced every 3 days, and cells were kept at a concentration no less than 1 × 106 cells mL−1. Human PB NK cells were expanded for 14–21 days prior to cell experiments. PB NK cells were purchased from StemCell Technologies. Cells were purified via FACS sorting using the surface marker CD56. All cell donors were ≥90% pure. SHSY5Y-GFP cells were cultured in RPMI 1640 (VWR, Radnor, PA) supplemented with 10% FBS (VWR, Radnor, PA) and 1× penicillin/streptomycin in T75 vented cell culture flasks. Cells were split every 2–3 days using trypsin (0.25%) (VWR, Radnor, PA).
5
Antibody-Dependent NK Cell Activation
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ELISA plates (NUNC, Thermo Fisher) were coated with HA antigens (H1 A/California/07/2009 and H3 A/Texas/50/2012), washed to remove unbound antigen, and blocked with 5% bovine serum albumin (Sigma) in PBS. Primary human NK cells were isolated from fresh buffy coats using the RosetteSep NK cell enrichment kit (StemCell) and rested overnight at 37C with 1 ng/ml IL-15 (StemCell). 1:25 diluted serum samples were added to the washed, HA-coated plates and incubated for 2 hours at 37C. Immediately prior to use, brefeldin A (Sigma), GolgiStop (BD), and fluorescent anti-CD107a (BD) were added to primary NK cells. Immune complexed plates were washed, and NK cells were added for 5 hours at 37C. After incubation, NK cells were removed and stained with fluorescent antibodies for cell surface markers CD3, CD56, and CD16 (BD). NK cells were fixed and permeabilized using Fixation & Permeabilization Media A & B (BD). Permeabilized NK cells were stained for intracellular makers MIP-1β and IFN-ɣ (BD). NK cells were quantified on the iQue Screener Plus using Forecyt software (Intellicyt). NK cells were defined as CD3 negative, CD16 positive. Each sample was assayed on two buffy coat donors.
6
SARS-CoV-2 S Protein-Induced NK Cell Responses
7
Human NK Cell Culture and Isolation
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The human NK92 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in α-minimal essential medium (Welgene) supplemented with 12.5% fetal bovine serum (FBS), 12.5% horse serum, 0.2 mM myo inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 1% antibiotics, and 20 ng/ml IL-2 in a humidified atmosphere with 5% CO2 at 37 °C. The human lung cancer lines (H358, H460, and H3122) were obtained from ATCC and cultured in RPMI medium supplemented with 10% FBS and 1% antibiotics. Primary NK cells were obtained from human cord blood provided by a local hospital (IRB approval number P01-201610-31-002), as described previously [29] (link). Briefly, CD3 cells and red blood cells were depleted from mononuclear cells with a Rosettesep cocktail (STEMCELL technologies) and CD3-depleted cells were cultured in α-minimal essential medium (Welgene) supplemented with human IL-15 (10 ng/mL), IL-21 (10 ng/mL), and 10−6 M hydrocortisone (StemCell Technologies).
8
Isolation and Activation of NK Cells for Coculture with MM
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NK cells were isolated from PB samples obtained from healthy volunteers. Undiluted PB was overlaid on a Ficoll density gradient and centrifuged at 200× g for 20 min without break. The platelet-enriched fraction was collected and stored at RT. The mononuclear cells were collected, and NK cells were isolated by negative depletion using the EasySep human NK cell isolation kit and EasySep magnet (Stemcell Technologies, Vancouver, BC, Canada), according to the manufacturer’s instructions. Isolated NK cells were cultured for 7 days in NK MACS medium (Miltenyi Biotech GmbH; Bergisch Gladbach, Germany) supplemented with human serum, IL-2 (500 U/mL,) and IL-15 (140 U/mL), both from Stemcell Technologies. Media was replenished every 3 days. MM cells were incubated with/without platelets and after incubation, cells were cocultured with/without platelet-autologous NK cells at different ratios for 5 h. Cell death was measured by flow cytometry using the Annexin V/7AAD assay (BD Biosciences).
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