CD4+CD25+ T cells and CD3−CD56+ NK cells were isolated from freshly-obtained PBMC by immunomagnetic sorting (Miltenyi, Germany) or by using a human NK cell Enrichment Kit (STEM Cell Technology), respectively, following the manufacturers’ instructions. Cell separation was performed using an AutoMACS (for Treg isolation) and EasySep magnet (for NK cell isolation) according to the manufacturer’s protocol. Flow-bases cell sorting was used to isolate NK cells from TIL. Viability of separated cells was measured using trypan blue dye.
Human nk cell enrichment kit
Manufactured by STEMCELL
Sourced in Germany
The Human NK Cell Enrichment Kit is a laboratory product designed to isolate natural killer (NK) cells from human peripheral blood or other sources. It utilizes a negative selection process to enrich for NK cells while depleting other cell types. The kit provides the necessary reagents and protocols to obtain a purified population of NK cells for further analysis or research applications.
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Lab products found in correlation
Rpmi 1640 media, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Immunomagnetic sorting, by Miltenyi Biotec (1 mentions)
Human serum, by Merck Group (1 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (1 mentions)
Human monocyte enrichment kit, by STEMCELL (1 mentions)
Human neutrophil enrichment kit, by STEMCELL (1 mentions)
Ficoll paque premium 1, by GE Healthcare (1 mentions)
Hetasep solution, by STEMCELL (1 mentions)
Cd45 pe, by BD (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by STEMCELL (1 mentions)
Il 18, by Thermo Fisher Scientific (1 mentions)
Il 12, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
7 aad, by BD (1 mentions)
Human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Human pan b cell enrichment kit, by STEMCELL (1 mentions)
7 protocols using human nk cell enrichment kit
1
Isolation of Immune Cell Subsets
2
NK Cell Cytotoxicity Assay Against Colorectal Cancer
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Frozen human peripheral CD56+ NK cells procured from ZenBio Inc. were thawed as per vendor recommended protocol in RPMI1640 media (GIBCO, Thermo Fisher Scientific) containing 10 ng/mL rh IL15 protein (R&D Systems Inc.), 1,000 units/mL rh IL2 protein (R&D Systems Inc.), 10% human serum (Sigma-Aldrich) and 1% pen-strep and incubated at 37°C and 5% CO2 incubator for approximately 48 hours. NK cells were also isolated from frozen as well as freshly isolated human PBMCs using human NK-cell enrichment kit (Stemcell Technologies Inc.) and EasySep magnet (Stemcell Technologies Inc.). Drug dilutions (4X), HCT116 cell suspension, and NK-cell suspension was prepared in assay media containing McCoy's 5A media, 1% FBS and 1% pen-strep. NK cells (effector cells) were plated on to a 96-well white clear-bottom plate (at a density of 50,000 cells/well) and cocultured with HCT116 cells (target cells) at a density of 5,000 cells/well (E:T = 10:1) in presence of TGFβ spiked at 1 ng/mL. Drugs were added to a final concentration of 56 nmol/L and incubated at 37°C, 5% CO2 for 72 hours. IFNγ levels were measured in the supernatant after approximately 72 hours as per manufacturer's instructions using human IFNγ Quantikine ELISA Kit. Absorbance readout was taken using a plate reader (BioTek Synergy HT or Spectramax M5e plate reader. Data were analyzed using GraphPad Prism software.
3
Cell Line and PBMC Cultivation Protocol
4
Isolation of Human Blood Immune Cells
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Human peripheral blood monocytes, neutrophils and NK cells were isolated from healthy male adult donor aphaeresis cones (National University Hospital, Blood Donation Centre, Singapore). This work was approved by the Institutional Review Board (IRB), NUS-IRB B-14-063E, National University of Singapore (NUS). Monocytes and NK cells were isolated as described previously [59 (link)]. Briefly, the buffy coat, which contained citrate-phosphate-dextrose (CPD) as anticoagulant, was diluted four times with PBS containing 2% FBS and 1 mM EDTA, and the mononuclear fraction was obtained via density gradient centrifugation with Ficoll-Paque Premium 1.073 (GE Healthcare). From the mononuclear fraction, the monocyte and NK cell populations were enriched with the Human Monocyte Enrichment Kit and Human NK Cell Enrichment Kit (Stemcell), respectively. For neutrophil isolations, buffy coats were diluted using Hank’s Balanced Salt Solution (HBSS; Life Technologies). Red blood cells were sedimented by gravity using Hetasep solution (Stemcell). Leukocytes were harvested and neutrophils were isolated using Human Neutrophil Enrichment kit (Stemcell).
5
Quantifying NK Cell Cytotoxicity against Breast Cancer Cells
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Lymphocytes were isolated from PBMCs with Ficoll-Paque Plus (StemCell- #07907) and NK cells were selected with a human NK cell enrichment kit (StemCell- #19055, >90% purity of CD56 + CD3- NK cells). The resultant NK cells were cultured for 16 hours in IL-2 (100 U/ml) or IL-12 (10ng/ml)/IL-15 (20ng/ml)/IL-18 (100 ng/ml)(Peprotech). Cells were washed 4X before being cultured with CFSE (5 μM) labelled MDA-231 (NCI-60 panel) cells at various E:T ratios (1:1, 5:1, 10:1) for 5 hours (MDA-231 cells alone = basal lysis). Cells were then stained with CD45-PE (BD Biosciences) antibody and immediately before flow cytometry, 7-AAD (BD, 5 μl/tube) was added to identify dead cells. FMO controls were included in each experiment. Flow cytometry analysis was performed to determine PE-CFSE + 7AAD+ cells and the above formula was applied.
6
Isolation of Lymphocyte Subsets from PBMC
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Lymphocyte subsets were purified from freshly prepared PBMC using EasySep (StemCell Technologies) negative magnetic selection kits: Human CD4+ T Cell Isolation Kit (StemCell Technologies, cat no. 17952), Human CD8+ T Cell Enrichment Kit (StemCell Technologies, cat no. 19053), Human NK Cell Enrichment Kit (StemCell Technologies, cat no. 19055) and Human Pan-B Cell Enrichment Kit (StemCell Technologies, cat no. 19554), per manufacturer’s instructions. After purification, 2 million (Pan B) or 3 million (CD4+ T, CD8+ T, and NK) isolated cells were lysed in Buffer RLT Plus (RNeasy Plus Mini Kit, Qiagen, cat no. 74134), per the manufacturer’s protocol, and lysates were kept at 4°C until RNA extraction. The remaining isolated cells were used for flow cytometric staining of markers to assess cell viability and purity as described below.
7
Quantifying NK Cell Proliferation and Cytotoxicity under TGFβ1 Influence
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NK cell proliferation and inhibition in the presence of TGFβ1 was determined as previously described.33 (link) Enriched NK cells from human PBMC (Human NK Cell Enrichment Kit, STEMCELL Technologies, 19055) were used. NK cells were activated by recombinant human IL-2 (10 IU/mL; R&D Systems, 202-IL/CF) for 3 days in the presence or absence of various concentrations of TGFβ1 (R&D systems). NK cell proliferation was observed by staining with Ki-67-BUV395 antibody (BD Bioscience, 564071) using flow cytometry. TGFβ1 was added at the time of setup with or without SAR439459 or isotype control (IgG4) for 3 days. NK cytotoxicity assays were performed using K562 cells (ATCC® CCL-243™) as targets. NK and K562 cells were cocultured (effector-to-target ratio 5:1) in RPMI-1640 media (GIBCO, 11835–030) supplemented with 10% FBS (GIBCO, 10082–147), and delivery of granzyme B was quantitated by intracellular FACS analysis using GranToxiLux™ assay kit as described (OncoImmunin, Inc, GTL702-8), for 2 hours. In some experiments, culture supernatants were collected for cytokine, GZB, perforin analysis by MSD (Meso Scale Diagnostics) or Luminex (ThermoFisher Scientific).
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