Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors by Ficoll density gradient centrifugation (PAN-Biotech, Germany). All blood donors gave their informed consent and all experiments were performed in accordance with relevant guidelines and regulations. Human NK cells were purified from PBMCs using the Dynabeads Untouched Human NK Cell kit (Thermo Fisher Scientific) according to manufacturer’s instructions. For NK cell activation and expansion, purified NK cells were cultured in 96-well round-bottom plates (Nunc) with irradiated K562-mbIL15-41BBL feeder cells (kind gift from Dario Campana) in IMDM Glutamax supplemented with 10% FCS and 1% penicillin/streptomycin, IL-2 (100 U/ml, NIH Cytokine Repository) and IL-15 (5 ng/ml, PAN-Biotech). Feeder cells were added at day 0 and day 7 of the culture and medium with fresh cytokines was exchanged every 2–3 days. IL-21 (100 ng/ml, Miltenyi Biotec) was added at the first day. NK cells were used for experiments between 3–6 weeks after isolation and they were between 90 and 99% CD3−, CD56+, and NKp46+ as assessed by flow cytometry.
Dynabeads untouched human nk cell kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dynabeads Untouched Human NK Cell kit is a magnetic bead-based cell separation system designed to isolate natural killer (NK) cells from human peripheral blood or leukapheresis samples. The kit utilizes a negative selection approach to enrich for NK cells without directly labeling the target cells, thereby preserving their native phenotype and functionality.
Automatically generated - may contain errors
Lab products found in correlation
Il 15, by PAN Biotech (4 mentions)
Il 21, by Miltenyi Biotec (4 mentions)
96 well round bottom plate, by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by PAN Biotech (3 mentions)
Ficoll density gradient centrifugation, by PAN Biotech (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions)
5 protocols using dynabeads untouched human nk cell kit
1
Isolation and Expansion of Human NK Cells
2
Isolation and Activation of Human NK Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll density gradient centrifugation (PAN-Biotech, Aidenbach Germany). Human NK cells were purified from PBMCs using the Dynabeads Untouched Human NK Cell kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer's instructions. For NK cell activation and expansion, purified NK cells were cultured in 96-well round-bottom plates (Nunc) with irradiated K562-mbIL15-41BBL (kind gift from Dario Campana) in IMDM Glutamax supplemented with 10% FCS and 1% penicillin/streptomycin, IL-2 (100 U/ ml, NIH Cytokine Repository) and IL-15 (5 ng/ml, PAN-Biotech). IL-21 (100 ng/ml, Miltenyi Biotec, Bergisch Gladbach Germany) was added at the first day. NK cells were between 90 and 99% CD3 -, CD56 + , and NKp46 + as assessed by flow cytometry. Huh7 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 4.5% glucose, 1% penicillin/streptomycin mixture and 10% heat-inactivated FCS. Primary human hepatocytes were cultivated in William's E medium (PAN Biotech, P04_29510) with 100 U/ml penicillin, 0,1 mg/ml streptomycin, 10 μg/ml gentamicin, 2 mM stable glutamine, 100 nM dexamethasone and 2 nM insulin-transferrin-selenite (ITS) supplement.
When plating cells, 10% fetal calf serum was added for the first 3-4 h of cultivation.
When plating cells, 10% fetal calf serum was added for the first 3-4 h of cultivation.
3
NK Cell-Mediated Suppression of ADE
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To investigate the function of NK cells in suppressing ADE, NK cells were depleted from whole PBMCs using the CD56+ magnetic bead kit (Miltenyi Biotec) according to the manufacturer’s protocol and the depletion achieved to >95%. ADE was set up with intact PBMCs and the NK-cell-depleted PBMCs and results were compared.
To further demonstrate the function of NK cells in suppressing ADE, we added the activated NK cells back to a new ADE assay set up with purified monocytes or PBMCs depleted of NK cells. Briefly, whole PBMCs were treated with serum only (for Control NK) or serum + virus (for Activated NK) as described previously for 24 hours. NK cells were then purified from the whole PBMC cultures using a Dynabeads Untouched Human NK Cell Kit (Thermo Fisher Scientific) and were added back to the new ADE assay. The new ADE assay was set up with either purified CD14 cells or with NK-depleted PBMCs. The cells were either uninfected (Mock), or infected with virus only (DENV), or infected with virus in the presence of ADE serum (ADE). The serum was not washed out. The NK cells were added at the beginning of the ADE cultures. Infection in monocytes was determined after 48 hours of ADE assay using the 2H2 monoclonal Ab. Infection was compared between cultures supplied with NK cells and cultures without NK cells.
To further demonstrate the function of NK cells in suppressing ADE, we added the activated NK cells back to a new ADE assay set up with purified monocytes or PBMCs depleted of NK cells. Briefly, whole PBMCs were treated with serum only (for Control NK) or serum + virus (for Activated NK) as described previously for 24 hours. NK cells were then purified from the whole PBMC cultures using a Dynabeads Untouched Human NK Cell Kit (Thermo Fisher Scientific) and were added back to the new ADE assay. The new ADE assay was set up with either purified CD14 cells or with NK-depleted PBMCs. The cells were either uninfected (Mock), or infected with virus only (DENV), or infected with virus in the presence of ADE serum (ADE). The serum was not washed out. The NK cells were added at the beginning of the ADE cultures. Infection in monocytes was determined after 48 hours of ADE assay using the 2H2 monoclonal Ab. Infection was compared between cultures supplied with NK cells and cultures without NK cells.
4
Isolation and Culture of Activated NK Cells
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NK cells were isolated and cultured as described [13 (link)]. In short, human NK cells were isolated from peripheral blood mononuclear cells with the Dynabeads® UntouchedTM Human NK Cell-Kit according to the manufacturer’s instructions (InvitrogenTM, Waltham, MA, USA). For experiments with resting NK cells, isolated NK cells were rested in IMDM medium (with GlutaMAXTM by Gibco (Waltham, MA, USA), 10% FCS, 1% penicillin/streptomycin) overnight and then used for experiments. To generate pre-activated NK cells, isolated NK cells were seeded in 96-well round-bottom plates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) at a density of 1.5–2 × 106 mL−1 with irradiated feeder cells (K562-mbIL15-41BBL) in medium with 200 U/mL IL-2 (National Institutes of Health Cytokine Repositor) and 100 ng/mL IL-21 (Miltenyi Biotec, Bergisch Gladbach, Germany). On day 8, NK cells were re-stimulated with fresh feeder cells. In the next weeks, NK cells were split on a density of 1.5–2 × 106 mL−1 in the presence of 100 U/mL IL-2. On day 14 recombinant 2.5 ng/mL IL-15 (PAN Biotech, Aidenbach, Germany) was added, and after three weeks NK cells could be used as pre-activated NK cells. In case of long-term treatment, Glutor (100 nM) or DMSO (0.1%) were added to the culture and every 72 h new substance was added.
5
Isolation and Activation of Human NK Cells
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Human NK cells were isolated from peripheral blood mononuclear cells with the Dynabeads ® Untouched TM Human NK Cell-Kit according to the manufacturer's instructions (Invitrogen). For experiments with resting NK cells, isolated NK cells were rested in IMDM medium (with GlutaMAX TM by Gibco, 10% FCS, 1% penicillin/streptomycin) overnight and then used for experiments. To generate pre-activated NK cells, isolated NK cells were seeded in 96-well round-bottom plates (Nunc) at a density of 1.5 -2*10 6 mL -1 with irradiated feeder cells (K562-mbIL15-41BBL) in a medium with 200 U/mL IL-2 (National Institutes of Health Cytokine Repositor) and 100 ng/mL IL-21 (Miltenyi Biotec). On day 8, NK cells were re-stimulated with fresh feeder cells. In the next weeks, NK cells were split at a density of 1.5-2 × 10 6 mL -1 , cultured in the presence of 100 U/mL IL-2 and 2.5 ng/mL IL-15 (PAN Biotech), and after three weeks NK cells were used as preactivated NK cells.
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