NK cytotoxicity assays were performed as previously described by Soland et al.16 (link) Briefly, MSC, nvMSC-HLA-G1 48 hours after transfection, and vMSC-HLA-G1 were plated in a flat-bottomed 96-well microplate (BD Falcon, Franklin Lakes, NJ) (1 × 105 cells/ml; 50 μl/well), and incubated in triplicate with different concentrations of NK-92 MI cells (ATCC, Rockville, MD) (20 × 105 cells/ml; 10 × 105 cells/ml; 5 × 105 cells/ml; 1 × 105 cells/ml; 2 × 104 cells/ml; 1 × 104 cells/ml; 50 μl/well) in α-MEM complete medium without phenol red (Gibco). NK-92MI cell line was used due to its robust proliferation rate in cell culture, and strong cytolytic activity towards several cells lines and primary cells without IL-2 supplementation. Furthermore, the NK-92 MI are also similar to activated NK cells with respect to their surface receptor expression.45 (link) The cytotoxicity tests were run at 20:1, 10:1, 5:1, 1:1, 0.2:1, and 0.1:1 E:T ratios following the guidelines of the CytoTox 96H Non-Radioactive Cytotoxicity Assay (Promega).
Nk 92mi cells
Manufactured by American Type Culture Collection
Sourced in United States
NK-92MI cells are a natural killer cell line derived from a 72-year-old male with non-Hodgkin's lymphoma. They are used as a model for natural killer cell research and function.
Automatically generated - may contain errors
Lab products found in correlation
α mem, by Thermo Fisher Scientific (4 mentions)
Folic acid, by Merck Group (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
Horse serum, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Cytotox 96h non radioactive cytotoxicity assay, by Promega (2 mentions)
Flat bottomed 96 well microplate, by BD (2 mentions)
α mem complete medium without phenol red, by Thermo Fisher Scientific (2 mentions)
K562 cell, by American Type Culture Collection (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Agm astrocyte growth medium bullet kit, by Lonza (1 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
E8 medium, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
21 protocols using nk 92mi cells
1
NK Cell Cytotoxicity Assay Protocol
2
Culturing Diverse Cell Lines for Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GSC lines, including MES28, GSC23, 456, 387, 4121 and 3691, were kindly provided by Dr. Jeremy N. Rich (UPMC). These cell lines were cultured in neurobasal medium (Gibco) supplemented with B27 (Life Technologies), bFGF and EGF (both 20 μg/ml, R&D Systems). NHAs were purchased from Lonza and cultured with an AGM™ Astrocyte Growth Medium Bullet Kit (Lonza) according to the manufacturer’s recommendation. NK-92MI cells (ATCC, CRL-2408) were cultured in NK complete medium (alpha minimum essential medium supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum and 12.5% foetal bovine serum (FBS) but without ribonucleosides and deoxyribonucleosides) as previously reported32 (link). 293T cells (ATCC, CRL-3216) and GL261 (DSMZ, ACC 802) were cultured in DMEM (Gibco) supplemented with 10% FBS according to standard protocols. The cell lines were authenticated using short tandem repeat (STR) fingerprinting method.
3
Optimizing NK Cell Cytotoxicity Assays
4
Stem Cell and Cell Line Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The experiments carried out in this project were in accordance with the National Institutes of Health Guidelines on Human Stem Cell Research. The protocols were approved by the University of Macau Panel of Research Ethics. H1 hESCs (Wicell, WA01) were maintained in monolayer on Matrigel (Corning, 354230) in E8 medium (Thermo, A1517001), 293T cells (ATCC, CRL-3216) in high glucose DMEM medium (Thermo, 11965092) with 10% FBS (Thermo, 16000044), K562 cells (ATCC, CCL-243) in RPMI1640 medium (Thermo, 11875093) with 10% FBS, NK-92MI cells (ATCC, CRL-2408) in αMEM (Thermo, 32561037) with inositol (Sigma, I5125, 0.2 mM), folic acid (Sigma, F8758, 0.02 mM), 2-mercaptoethanol (Thermo, 21985023, 0.1 mM), 12.5% horse serum (Thermo, 26050088) and 12.5% FBS, and MSCs in αMEM (Thermo, 12571063) with 20% FBS.
5
Culturing HNSCC and NK92MI Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All HNSCC cell lines were cultured and expanded in T‐25 flasks (ThermoFisher) using RPMI 1640 medium enriched with 10% Fetal Bovine Serum (HyClone, USA) and 1% Penicillin Streptomycin (ThermoFisher). Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. Medium was changed every 2–3 days and the cells were passaged when reaching about 80% of confluency (~every 3 days). NK92MI cells (ATCC, USA) were cultured and expanded in T‐25 flasks (ThermoFisher) using alpha‐Minimum Essentials Eagle Medium (MEM) enriched with 12.5% FBS (HyClone, USA), 12% horse serum (ThermoFisher), 4% MEM vitamin solution (ThermoFisher) and 0.1 mM beta‐mercaptoethanol (Thermofisher).
6
Orthotopic Xenograft Model for NK Immunotherapy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An orthotopic xenograft model was established in this study as previously described17 (link),52 . Mice were randomly allocated to treatment or control groups. Animals were first anaesthetized with isoflurane. CircEZH2 stable KD or control GSCs (2.5 × 104) or GL261 cells (5.0 × 104) in 5 μl of PBS were then intracranially injected using a 10-μl Hamilton syringe through a guide screw into the right frontal lobe at a depth of 3 mm. Ten days after inoculation, 2 × 106 NK-92MI cells (CRL-2408, ATCC) or primary murine NK cells in 5 μl of PBS were injected intracranially every 7 or 5 days via the same guide screw at the same depth. For the GL261 model, the mice also received an intraperitoneal injection of anti-PD1 antibody (BE0146, BioXcell, 10 mg/kg, 1:5 diluted in PBS) or PBS on the same day as the intracranial injection of NK cells. The mice were observed and weighed daily, and humanely euthanized once they presented neurological symptoms, displayed 20% weight loss or became moribund, in order to ensure that the intracranial tumour burden would not exceed the ethical limits. This was permitted by the Ethics Institutional Review Boards of the First Affiliated Hospital of Sun Yat-sen University.
7
Establishment of AR knockdown cell lines
8
Culture of NK-92MI and HT29 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK-92MI cells (ATCC) were cultured in
Minimum Essential Medium α without nucleosides (Gibco), supplemented
with 12.5% total volume fetal calf serum (HyClone), 12.5% total volume
horse serum (Stem Cell Technologies), 2 mM Glutamax (Gibco), 1×
penicillin–streptomycin (Gibco), 0.02 mM folic acid (Sigma-Aldrich),
0.2 mM myo-inositol (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol
(Sigma-Aldrich) at 37°C, 5% CO2. In addition, 50 U
recombinant human IL2 (PeproTech) was supplemented to the culture
medium during the first 5 days after starting cell culture.
Subculture of the cells was performed every 2–3 days. Viable
cell clusters were collected by centrifugation at 175g for 5 min, after which the cells were split in a 1:4 ratio to achieve
2–3 × 105 cells/mL in fresh NK medium.
HT29 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco)
and 20 mM HEPES at 37 °C, 5% CO2.
Minimum Essential Medium α without nucleosides (Gibco), supplemented
with 12.5% total volume fetal calf serum (HyClone), 12.5% total volume
horse serum (Stem Cell Technologies), 2 mM Glutamax (Gibco), 1×
penicillin–streptomycin (Gibco), 0.02 mM folic acid (Sigma-Aldrich),
0.2 mM myo-inositol (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol
(Sigma-Aldrich) at 37°C, 5% CO2. In addition, 50 U
recombinant human IL2 (PeproTech) was supplemented to the culture
medium during the first 5 days after starting cell culture.
Subculture of the cells was performed every 2–3 days. Viable
cell clusters were collected by centrifugation at 175g for 5 min, after which the cells were split in a 1:4 ratio to achieve
2–3 × 105 cells/mL in fresh NK medium.
HT29 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Gibco)
and 20 mM HEPES at 37 °C, 5% CO2.
9
Isolation and Culture of NK92-MI Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NK92-MI cells (ATCC CRL-2408), were grown and cultivated in minimum essential medium (MEM) alpha medium (Life Technologies, Carlsbad, CA) supplemented with 12.5% FBS and donor horse serum (Atlanta Biologicals, Lawrenceville, GA), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, and 0.02 mM folic acid. All cells were cultured at 37°C in a humidified 5% CO2/95% air environment. Peripheral blood mononuclear cells (PBMCs) were isolated from ethylene-diamine-tetra-acetic acid (EDTA)-treated whole-blood samples by Histopaque-1077 (Sigma Chemicals, St. Louis, MO) density gradient centrifugation using LeucoSep tubes (Greiner, Monroe, NC) from healthy individuals with prior approval from the Office of Protection of Human Subjects, The North Texas Regional Institutional Review Board (IRB# 20–28) of UNT Health Science Center, Fort Worth, TX. A written consent was obtained from healthy donors and no minors were involved in this study. Primary NK cells were isolated from the PBMCs using NK isolation kit (Miltenyi Biotec, San Diego, CA) and the purity was determined by flow cytometry using anti-human CD56 mAb.
10
Cell Culture Protocols for Colorectal and Renal Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five human colorectal cell lines including SW480, SW620, HCT116, HT-29, and CACO-2 and the human renal epithelial cell line 293 T (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China) were cultured in DMEM/F12 with 10% fetal bovine serum (FBS). For passaging, cells were digested with 0.25% trypsin and collected after centrifugation at 1050 × g for 5 min. Cells were then passaged at a ratio of 1:3 to 1:6. NK-92® MI cells (ATCC®, CRI 2408TM) were cultured in MEMα with 10% horse serum, 10% FBS, 0.1 mM β-mercaptoethanol, and 1% penicillin–streptomycin. For passaging, NK-92® MI cells as a suspension culture were mixed with medium and then were collected by centrifuging as above, followed by passaging at a ratio of 1:2.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!