The cell lines MDA-MB-231/GFP (Cell Biolabs) and NIH 3T3 (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin-streptomycin. The NK-92 cells (ATCC) were cultured in Alpha minimum essential medium (ATCC, VA, USA) supplemented with 0.2 mM inositol (Sigma), 0.1 mM 2-mercaptoethanol (Sigma), 0.02 mM folic acid (Sigma), 100–200 U/ml recombinant interleukin-2 (PeproTech, NJ, USA), 12.5% horse serum, 12.5% FBS, and 1% penicillin-streptomycin. K562 cells (ATCC) were cultured in Iscove’s modified Dulbecco’s medium, supplemented with 10% (v/v) FBS and 1% penicillin-streptomycin. All cells were grown in a humidified atmosphere of 5% (v/v) CO2 at 37°C. Adherent cells (MDA-MB-231 and NIH 3T3) at 80% confluence in a 60 mm × 15 mm Petri dish were harvested by trypsin digestion and dispensed into a cell suspension with various concentrations according to the experimental requirement. Suspended cells (NK-92 and K562) were centrifuged and washed by cell-free media or PBS and finally adjusted into known cell centration.
Nk 92 cells
Manufactured by American Type Culture Collection
Sourced in United States
NK-92 cells are a natural killer (NK) cell line derived from the peripheral blood of a patient with non-Hodgkin's lymphoma. They are an immortalized cell line that can be used for research and experimental purposes. NK-92 cells exhibit characteristics of mature and activated NK cells, and they can be used as a model system to study NK cell biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Horse serum, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Folic acid, by Merck Group (6 mentions)
Myo inositol, by Merck Group (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
2 mercaptoethanol, by Merck Group (3 mentions)
Mcf 7, by American Type Culture Collection (3 mentions)
Mda mb 231, by American Type Culture Collection (3 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (3 mentions)
Inositol, by Merck Group (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions)
Nk92 cells, by Thermo Fisher Scientific (3 mentions)
Mem without ribonucleosides media, by Thermo Fisher Scientific (2 mentions)
Dmem f12 media, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Hydrocortisone, by Merck Group (2 mentions)
29 protocols using nk 92 cells
1
Cell Culture Protocols for Cancer and Immune Cell Lines
2
Culturing Human NK92 Cell Line
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For in vitro studies, NK92 cells (CRL-2407, human NK line) were obtained from ATCC. The cells were cultured in a minimum essential medium (MEM, Sigma, St. Louis, MO, USA) containing 12.5% fetal bovine serum (HyClone, Logan, UT, USA), 12.5% horse serum (HyClone), 200 U recombinant IL-2/mL (Invitrogen, Carlsbad, CA, USA), 2 mmol/L L-glutamine, 0.2 mmol/L myoinositol, 0.1 mmol/L 2-mercaptoethanol, and 0.02 mmol/L folic acid (all Sigma, St. Louis, MO, USA) at 37°C in a 5% CO2 incubator. Fresh medium was replaced every 2 d; cells reached confluence at 5 d of culture.
3
Expansion of NK-92 Immune Cells
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NK-92 cells (purchase from ATCC CRL-2407) are cultured for 3 weeks prior to measurements in Alpha-MEM medium (Stemcell Technologies) with 15% fetal calf serum, 15% horse serum, 500 IU/ml human IL2-cytokine, and 1% penicillin–streptomycin solution (10,000 units/ml penicillin, 10,000 µg/ml streptomcycin).
4
Analyzing Immune Cell Killing of Cancer Cells
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The following human cancer cell lines were used in live cell imaging to assess immune cell killing of tumor cells: MDA-MB-231 (HTB-26), triple-negative (i.e., ER−PR−HER2−) epithelial breast carcinoma; NCI-H460 (HTB-177), large cell lung carcinoma; and LNCaP (HTB-1740), androgen-sensitive prostate adenocarcinoma. Cell lines were purchased from the ATCC and were cultured in reduced-riboflavin conditions using CMRL-1066 (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (HyClone) and 1% antibiotic-antimycotic solution (Gibco Life Technologies). Cells were maintained at 37°C in 5% CO2, and adherent cells were detached with trypsin (0.25%-EDTA, HyClone) for passaging and further culture. NK-92 cells were also purchased from ATCC and maintained in MEM-α containing 12.5% FBS, 12.5% horse serum (Gibco Life Technologies), 100 IU/mL IL-2 (R&D Systems), 0.2 mM inositol (Sigma-Aldrich), 0.02 mM folic acid (Sigma-Aldrich), and 0.1 mM mercaptoethanol (Gibco Life Technologies). Other cancer cell lines were screened for expression of CD318 (Table 1 ) and were grown in RPMI-1640 (HyClone) supplemented with 10% heat-inactivated FBS (HyClone) and 1% antibiotic-antimycotic solution (Gibco Life Technologies).
5
Comparison of Immune Cell Lines for Research
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RMA, RMA-S, EL4, EL4H60 and YAC-1 cells were described previously [27] (link). CCRF-CEM, human acute T lymphocytic leukemia cell line was kindly provided by Dr. Matthew Janes (Intellikine, La Jolla, CA). The human NK cell line NKL was provided by Dr. Melissa Lodoen (UC Irvine). K562 cells were obtained from Dr. Tiong Ong (Duke-National University of Singapore). NK92 cells were from ATCC (Manassas, VA). All cells were cultured in RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin-streptomycin, 2 mM L-glutamine, 5 mM HEPES buffer, and 50 mM 2- mercaptoethanol, which we refer to as complete RPMI. For NKL and NK92 cells, the media was supplemented with human IL-2 (500 U/ml).
6
Cell Culture Maintenance Protocols
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NK-92 cells (directly purchased from ATCC) were maintained in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 400 U/mL rhIL-2, and 0.1 mM 2-mercaptoethanol. GBM43 and GBM10 cells (kindly provided by Dr. Karen E. Pollok, Indiana University School of Medicine) were grown in DMEM supplemented with 10% FBS and 1% HEPES. All cell lines were incubated at 37 °C in a humidified 5% CO2 environment. A549 cells (kindly provided by Dr. Darci Trader, Purdue University) were grown in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. PC3 cells (kindly provided by Dr. Marxa L. Figueiredo, Purdue University) were grown in RPMI1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
7
Cell Lines for AQP4 and NK Cell Studies
8
Breast Cancer Cell Line Activation and Co-culture
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This study was approved by the Xiangya Hospital of Central South University (ethics number: 202109924). Female patients with breast cancer (n = 58) were recruited in this study. Written consents were obtained from all participants. The clinicopathological characteristics of breast cancer patients, including age, menopause, tumor size, lymph node metastasis and TNM stage, were listed in Table S1 .
All methods and protocols were performed in accordance with Practical Guide for Animal Experiment Management and Operation. Female NOD-SCID mice (4 ∼ 6-wk-old) were used in this study. All mice have free access to standard chow and water under controlled temperature and constant 12h/12h light/dark cycles.
Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA). MCF10A cells were cultured in F12/DMEM (Gibco, Grand Island, NY, USA). The breast cancer cells were grown in DMEM (Gibco) containing 10% FBS. NK92 cells were cultured in MEMα containing 12.5% FBS, 2 mM L-glutamine and 12.5% horse serum (Gibco). For NK92 cell activation, cells were stimulated with 100 U/mL IL-2 (Gibco, PHC0023) for 24 h. For co-culture, the activated NK-92 cells were co-cultured with MCF-7 or MDA-MB-231 cells at a ratio of 10:1 for 4 h. All cells were maintained at 37°C/5% CO2.
All methods and protocols were performed in accordance with Practical Guide for Animal Experiment Management and Operation. Female NOD-SCID mice (4 ∼ 6-wk-old) were used in this study. All mice have free access to standard chow and water under controlled temperature and constant 12h/12h light/dark cycles.
Human mammary epithelial cell line MCF10A and human breast cancer cell lines MCF-7, Hs578T, T47D, MDA-MB-231, MDA-MB-468 cells and human NK cell line NK92 cells were from ATCC (Manassas, VA, USA). MCF10A cells were cultured in F12/DMEM (Gibco, Grand Island, NY, USA). The breast cancer cells were grown in DMEM (Gibco) containing 10% FBS. NK92 cells were cultured in MEMα containing 12.5% FBS, 2 mM L-glutamine and 12.5% horse serum (Gibco). For NK92 cell activation, cells were stimulated with 100 U/mL IL-2 (Gibco, PHC0023) for 24 h. For co-culture, the activated NK-92 cells were co-cultured with MCF-7 or MDA-MB-231 cells at a ratio of 10:1 for 4 h. All cells were maintained at 37°C/5% CO2.
9
Engineered CD16 Expression in Cell Lines
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HEK293 cells (a human embryonic kidney cell line) and NK92 cells (a human NK cell line) (ATCC, Manassas, VA) were cultured per the company’s instructions. HEK293 cells were transiently transfected with pcDNA3.1 with or without CD16b, CD16b/S197P, and/or L-selectin using Lipofectamine 2000 (Invitrogen) per the manufacturer’s instructions. HEK293 cells stably expressing human FcRγ were transiently transfected with pcDNA3.1 with or without CD16a or CD16a/S197P by the same approach. NK92 cells were stably transduced with pBMN-IRES-EGFP with or without CD16a or CD16a/S197P by retrovirus generation and infection procedures described previously [26 (link)–28 (link)]. Construct expression was assessed by EGFP fluorescence and CD16 staining, as determined by flow cytometry. Human iPSCs (UCBiPS7, derived from umbilical cord blood CD34 cells) were maintained on mouse embryonic fibroblasts [29 (link), 30 (link)]. Stable expression of CD16a or CD16a/S197P was performed using a Sleeping Beauty transposon system, as previously described [23 (link), 24 (link)]. Briefly, iPSCs were nucleofected with pKT2-IRES-GFP:zeo in combination with transposase DNA in nucleofector solution V (Lonza Inc., Gaithersburg, MD) using program setting B16. Nucleofected cells were immediately suspended in iPSC growth medium containing zeocin (50μg/ml) and seeded onto mouse embryonic fibroblasts.
10
Breast Cancer and NK-92 Cell Culture
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MDA-MB231 (ATCC), MCF7 (ATCC) and bone metastatic BoM-1833 breast cancer cell lines (kindly provided by Joan Massague) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals) and 1× penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2.
NK-92 cells (ATCC; CRL-2407) were cultured in Minimum Essential Medium α ( -MEM) without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/mL recombinant human IL-2 (Pepro Tech), and 1× penicillin/streptomycin at 37°C in 5% CO2.A clonal MCF10A cell line expressing Muc1-GFP was generated previously and referred to here as 1E7 cells[41 (link)]. The cells were cultured in DMEM/F12 media (Thermo Fisher Scientific) supplemented with 5% horse serum (Thermo Fisher Scientific), 20 ng/mL EGF (Pepro Tech), 10 mg/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma) and 1x penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2.
NK-92 cells (ATCC; CRL-2407) were cultured in Minimum Essential Medium α ( -MEM) without ribonucleosides media (Thermo Fisher Scientific) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.2 mM Myo-inositol (Sigma Aldrich), 0.1 mM 2-mercaptoethanol (Thermo Fisher Scientific), 0.02 mM folic acid (Millipore Sigma), 100 U/mL recombinant human IL-2 (Pepro Tech), and 1× penicillin/streptomycin at 37°C in 5% CO2.A clonal MCF10A cell line expressing Muc1-GFP was generated previously and referred to here as 1E7 cells[41 (link)]. The cells were cultured in DMEM/F12 media (Thermo Fisher Scientific) supplemented with 5% horse serum (Thermo Fisher Scientific), 20 ng/mL EGF (Pepro Tech), 10 mg/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma) and 1x penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2.
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