Validation for cell sorting and transfection was done with D1 cells from ATCC (ATCC® CRL-12424™), a commercially available mouse bone marrow precursor cell line. These cells were not independently authenticated since they were obtained directly from ATCC. Cells were tested for mycoplasma using the PlasmoTestTM – Mycoplasma Detection Kit from InvivoGen (Cat # rep-pt2) and PCR analysis. All cells were confirmed to be free of mycoplasma.
Plasmotest mycoplasma detection kit
Manufactured by InvivoGen
Sourced in United States, France, China
The PlasmoTest Mycoplasma Detection Kit is a laboratory tool designed to detect the presence of mycoplasma contamination in cell cultures. It provides a reliable and efficient method for monitoring mycoplasma levels in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (19 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (15 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (13 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Penicillin, by Thermo Fisher Scientific (7 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Mda mb 231, by American Type Culture Collection (5 mentions)
Hek293, by American Type Culture Collection (4 mentions)
Rwpe 1, by American Type Culture Collection (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
Penicillin, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Glutamax supplement, by Thermo Fisher Scientific (3 mentions)
Hsc 3, by Japanese Collection of Research Bioresources Cell Bank (3 mentions)
Hepes, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (3 mentions)
Lncap, by American Type Culture Collection (3 mentions)
90 protocols using plasmotest mycoplasma detection kit
1
Cell Validation and Mycoplasma Testing
2
Cell Culture Conditions and Treatments
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Human epithelial Cell (BEAS2B), lung squamous carcinoma (H520, H2170), small cell lung cancer (SBC5, H69), and HEK293 cells were used in this study and were cultured in RPMI 1640 (Corning, Manassas, VA, USA) supplemented with 10% fetal bovine serum (R&D, Cat #: S11550H), 1% of L-glutamine (Corning, Cat #: 25-005-CI), penicillin/streptomycin (Corning, Cat #:30002312), sodium bicarbonate (Corning, Cat #:19718005) and sodium pyruvate (Corning, Cat #:32919014). The cells were cultured at 37°C, 20%O2, and 5% CO2 conditions. All cells were tested for mycoplasma using the PlasmoTestTM Mycoplasma Detection kit (InvivoGen, Cat #: rep-mys-50, San Diego, CA, USA). Cisplatin was purchased from Sigma-Aldrich (Cat #:1134357-100MG) and dissolved in H2O. Carfilzomib (CFZ) and Ixazomib (IXA) were purchased from Selleck Chemicals (Cat #: S2853 and Cat #: S2180, respectively, Houston, TX, USA) and were dissolved in DMSO (ATCC, Cat #: 67-68-5). ActD was purchased from Sigma-Aldrich (Cat #: A1410-5mg). DMSO was used as vehicle control.
3
Culturing Murine TNBC Cell Lines
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Firefly luciferase-expressing 4T1 mammary tumor cells that resemble metastatic human TNBC were provided as a kind gift from Prof. Clare Isacke (Breakthrough Breast Cancer Research Center, London, UK) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin (both from Sigma-Aldrich, Overijse, Belgium) at 37°C and 5% CO2. Py230 mammary tumor cells, derived from the American Type Culture Collection (ATCC) and reported to model TNBC,18 (link),20 (link) were cultured in Ham’s F-12 K Medium (Thermo Fisher Scientific) supplemented with 5% heat-inactivated FBS (Thermo Fisher Scientific), 0.1% MITO+ Serum Extender (Corning, New York, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich) at 37°C and 5% CO2. Checks for mycoplasma and other bacterial contamination were routinely performed using a PlasmoTestTM mycoplasma detection kit (Invivogen, San Diego, USA). Harvesting of the cells involved washing with phosphate buffered saline (PBS) and incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) for 5 minutes (min). The cells were subsequently centrifuged at 805 × g for 5 min, resuspended in PBS and counted with a Bürker chamber.
4
Cell Culture and TGFβ Treatment
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MCF10A cells were a gift from Dr. Sendurai Mani and were cultured as previously described [46 (link)]. HMLE cell lines were also a gift from Dr. Sendurai Mani and were cultured as described previously [43 (link)]. Cells were plated at 10,000 cells/cm2 and passaged every other day to maintain consistent densities. For TGFβ treatment, media was supplemented with 5 ng/mL recombinant human TGFβ-1 (R&D Systems, Minneapolis, MN, USA; resuspended in 4 mM HCl, 0.1% BSA) and were replenished with every cell passage. MCF7, SKBR3, MDA-MB-231, Hs-578t cell lines (ATCC, Manassas, VA, USA), and HEK-293T cells, which were a gift from Dr. Jason Herschkowitz, were cultured in DMEM media (Corning, Corning, NY, USA) supplemented with 10% FBS (Cytiva, Marlborough, MA, USA), and 1% penicillin/streptomycin (Lonza, Basel, Switzerland). Cells were regularly tested for mycoplasma contamination using PlasmoTestTM Mycoplasma Detection Kit (InvivoGen, San Diego, CA, USA).
5
Cell Line Characterization and Validation
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All cell lines were passaged in αMEM supplemented with 5% fetal bovine serum (FBS), and insulin/transferrin/selenium supplement (Roche, Basel, Switzerland) without antibiotics for less than 3 months from frozen stocks confirmed to be mycoplasma-free. The human breast cancer estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 cell lines, and the human embryonic kidney cell line HEK293 were purchased from the American Type Culture Collection (ATCC). The Chinese hamster ovary CHO cell lines 51D1 and 51D1.3 were obtained from Prof. William Wilson. Their DNA repair genotypes and origins, were as follows: 51D1 [36 (link)] (rad51d knockout) and 51D1.3 [36 (link)] (51D1 complemented with CHO Rad51d). All cell lines tested negative for mycoplasma contamination (PlasmoTestTM—Mycoplasma Detection kit, InvivoGen, San Diego, CA, USA).
6
Characterization of Cell Lines for Preclinical Research
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Human ALL NALM6 cells (ATCC, #CRL-3273) and human lymphoma EL4 cells (ATCC, #TIB-39) were authenticated by STR profiling (University of Arizona Genetics Core or Bio-Synthesis Inc.), and routinely tested for Mycoplasma spp. contamination with the PlasmoTestTM Mycoplasma Detection Kit (InvivoGen, #rep-pt1). NALM6 cells were transduced to stably express luciferase and GFP (NALM6-BLIV cells) with the MSCV-GFP-T2A-Luciferase Minicircle (System Biosciences, #BLIV301PA-1) and the calcium phosphate method [14 (link)]. All cancer cell lines were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Life Technologies, #12440061) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies, #10438026) and 100 U/mL penicillin-streptomycin (Life Technologies, #15140122). T cells were cultured in RPMI-1640 medium (Life Technologies, #11875119) supplemented with 10% heat-inactivated FBS.
7
Extraction and Fractionation of Cellular Proteins from ER+ Breast Cancer Cells
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Epithelial estrogen receptor positive (ER+) MCF7 breast cancer cells (ATCC HTB-22, Manassas, Virginia) were cultured in Dulbecco’s Modified Eagle Medium (Corning, Corning, NY) supplemented with 10 % fetal bovine serum (Gibco, Billings, MT) and 1 % penicillin/streptomycin (Gibco, Billings, MT). Cells were regularly tested for mycoplasma contamination using PlasmoTestTM Mycoplasma Detection Kit (InvivoGen, San Diego, CA) and passaged when 80 % confluent.
Whole-cell protein was harvested using the Pierce Co-Immunoprecipitation (Co-IP) Kit (ThermoFisher Scientific, Waltham, MA) and subcellular protein fractionation was performed on 1.0 x 107 cells using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific, Waltham, MA). Protein concentrations were determined using bicinchoninic acid (BCA) assays28 (link) and subcellular fractionation was confirmed by immunoblotting with established protein markers for each cellular fraction (Figure S1 and Table S1 ).
Whole-cell protein was harvested using the Pierce Co-Immunoprecipitation (Co-IP) Kit (ThermoFisher Scientific, Waltham, MA) and subcellular protein fractionation was performed on 1.0 x 107 cells using the Subcellular Protein Fractionation Kit for Cultured Cells (ThermoFisher Scientific, Waltham, MA). Protein concentrations were determined using bicinchoninic acid (BCA) assays28 (link) and subcellular fractionation was confirmed by immunoblotting with established protein markers for each cellular fraction (
8
Cell Line Sources and Culture Conditions
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DAOY and D283 cell lines were obtained from ATCC (Manassas, USA), ONS76 from Annette Künkele (Charité Universitätsmedizin, Berlin, Germany), UW-228-3 and D458 from John R. Silber (University of Washington, Seattle, USA), HD-MB03 from Till Milde (DKFZ, Heidelberg, Germany) and CHLA-01 and CHLA-01R from Geoff Pilkington (University of Portsmouth, UK). DAOY, D283, D425, and D458 cells were cultured in DMEM with 10% foetal bovine serum (FBS, HyClone (Logan, Utah, USA), SH30541.03). UW-228-3 cells in DMEM/F-12 with 15% FBS and 1% sodium pyruvate. ONS76 and HD-MB03 cells were cultured in RPMI 1640 with 10% FBS. CHLA-01 and CHLA-01R were cultured in DMEM/F-12 supplemented with 2% B-27 (Gibco (Loughborough, UK), 17504044), 20 ng/mL recombinant human epidermal growth factor (EGF, Gibco, PHG0315) and 20 ng/mL recombinant human basic fibroblast growth factor (bFGF, Gibco, PHG0266). All cell lines were grown under antibiotic-free culture conditions at 5% CO2 and 37 °C. Mycoplasma testing was performed monthly using a PlasmoTestTM Mycoplasma Detection kit (InvivoGen (San Diego, CA, USA); rep-pt1) as per the manufacturer’s instructions.
9
Culturing Metastatic Breast Cancer Cell Lines
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Firefly luciferase-expressing 4T1 (4T1-luc) mammary tumor cells which resemble metastatic human TNBC were provided as a kind gift from Prof. Clare Isacke (Breakthrough Breast Cancer Research Centre, London, UK). The 4T1-luc cells were cultured as previously described [22 (link)]. Firefly luciferase-expressing 66cl4 (66cl4-luc) cells were kindly provided by Prof. Traci R. Lyons (University of Colorado Anschutz Medical Campus, Denver, CO, USA). The 66cl4-luc cells were cultured using Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 100U/ml penicillin, 100 µg/ml streptomycin (both from Sigma-Aldrich, Overijse, Belgium), 1% Gibco™ MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific) and 200 µg/ml hygromycin B (Thermo Fisher Scientific) at 37 °C and 5% CO2. Both cell lines were routinely checked for mycoplasma and other bacterial contamination using a PlasmoTestTM mycoplasma detection kit (Invivogen, San Diego, USA). Harvesting of confluent cells was performed through incubation with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) for 5 min and centrifugation at 400 × g for 5 min for subsequent counting with a Bürker chamber.
10
Cholangiocarcinoma Cell Line Cultivation
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