Natural killer (NK) cells were prepared from freshly collected plasma apheresis peripheral blood obtained from healthy donors at the Life Stream Blood Bank (San Bernardino, CA, USA), in accordance with Loma Linda University IRB protocols. Peripheral blood was prepared as described previously [47 (link)]. NK cells were obtained from the total lymphocyte fraction by negative immunomagnetic cell separation using either EasySep or RosetteSep negative selection kit (STEMCELL, Vancouver, BC, USA) or MACS magnetic NK cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). The purity of the NK cell population was determined by flow cytometric analysis and ranged from 85–95%, with all kits showing comparable performance in obtaining CD3-/CD56+ NK cells (greater than 90% purity and viability). NK cells were cultured at a high density of 5 × 106 cells/well in 6-well non-pyrogenic polystyrene culture plates overnight in RPMI 1640 media (Mediatech Inc. Manassas, VA, USA), supplemented with 10% FBS (Hyclone, Logan, UT, USA), 1 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 100 U/mL human recombinant IL-2 before use in experiments.
Rosettesep negative selection kit
Manufactured by STEMCELL
Sourced in United States
The RosetteSep negative selection kit is a cell isolation product designed to enrich specific cell types from heterogeneous cell populations. It utilizes a tetrameric antibody cocktail that binds to unwanted cells, facilitating their removal through density-based centrifugation. The core function of this kit is to provide a straightforward method for the isolation of desired target cells from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions)
Cd16 apc cy7, by BD (2 mentions)
Cd3 alexa fluor 700, by BD (2 mentions)
Recombinant il 15, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Golgistop, by BD (2 mentions)
Rpmi 1640 media, by Corning (1 mentions)
Fludarabine, by Merck Group (1 mentions)
Fludarabine, by Santa Cruz Biotechnology (1 mentions)
Mafosfamide, by Santa Cruz Biotechnology (1 mentions)
Ficoll plaque plus, by Cytiva (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Cd56 pe cy7, by BD (1 mentions)
Bdbv gp, by IBT Bioservices (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Ebov gp, by IBT Bioservices (1 mentions)
4 protocols using rosettesep negative selection kit
1
Isolation and Expansion of Human NK Cells
2
CLL Cell Isolation and DNA Damage Assay
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Peripheral blood was obtained from CLL patients with written informed consent in accordance with the Declaration of Helsinki and under protocols approved by the Institutional Review Board of the Ohio State University. CLL cells and normal B-cells were isolated using ficoll density gradient centrifugation (Ficoll-Plaque Plus; Amersham Biosciences) and enriched for B-cells using the Rosette-Sep negative selection kit (StemCell Technologies) according to manufacturer protocol. Cryopreserved cells used in the two prognostic datasets were obtained from the CLL Research Consortium (CRC) tissue bank or from the ECOG-2997 clinical trial. Cells were thawed in RPMI 1640 media then washed in PBS to obtain cell pellets.
The OSU-CLL cell line was grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2mM L-glutamine (Invitrogen), 100U/mL penicillin, and 100ug/mL streptomycin (Sigma). For DNA damage experiments, 2×106 cells were incubated with vehicle (DMSO), 10uM fludarabine (Sigma), 2.36uM mafosfamide (Santa Cruz), or 10uM fludarabine plus 2.36uM mafosfamide. All conditions were given equivalent volumes of DMSO.
The OSU-CLL cell line was grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2mM L-glutamine (Invitrogen), 100U/mL penicillin, and 100ug/mL streptomycin (Sigma). For DNA damage experiments, 2×106 cells were incubated with vehicle (DMSO), 10uM fludarabine (Sigma), 2.36uM mafosfamide (Santa Cruz), or 10uM fludarabine plus 2.36uM mafosfamide. All conditions were given equivalent volumes of DMSO.
3
NK Cell-Mediated BDBV GP Antibody Response
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Human NK cells were enriched from peripheral blood by negative selection using RosetteSep negative selection kit (Stem Cell Technologies) followed by Ficoll separation. NK cells were rested overnight in the presence of 1 ng/ml recombinant IL-15 (PeproTech). 3 μg/ml of BDBV GP (IBT Bioservices) was coated on a Maxisorp ELISA plate (Nunc) at 4°C overnight, and plates were blocked with 5% BSA prior to addition of antibodies (5 μg/ml) in PBS for 2 hours at 37°C. The control EBOV-specific mAb c13C6 was purchased from IBT Bioservices. Unbound antibodies were removed by washing wells 3X with PBS prior to addition of NK cells. The NK cells were added at 5 x 104 cells/well in the presence of brefeldin A (Sigma Aldrich), GolgiStop (BD Biosciences), and anti-CD107a PE-Cy5 antibody (BD Biosciences clone H4A3) and incubated for 5 hours at 37°C. NK cells were stained with flow cytometry antibodies for the following surface markers: CD3 AlexaFluor700 (BD Biosciences clone UCHT1), CD56 Pe-Cy7 (BD Biosciences clone B159), and CD16 APC-Cy7 (BD Biosciences clone 3G8), followed by intracellular staining for IFNγ (FITC, BD Biosciences clone B27) and MIP-1β (PE, BD Biosciences clone D21-1351) to detect the production of cytokines and chemokines. Cells were analyzed by flow cytometry on a BD LSRII flow cytometer and data was analyzed using FlowJo software.
4
Antibody-Mediated NK Cell Activation
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