The human epithelial cell lines HeLa (human cervical cancer), MCF-7 (human breast cancer), and HepG2 (human hepatocarcinoma) were obtained from American Type Culture Collection (Rockville, MD, USA). MCF-7 cells were grown in DMEM while HeLa and HepG2 cells were grown in RPMI (VWR International, Radnor, PA, USA). Both culture medium were supplemented with 5% FBS (VWR International, Radnor, PA, USA), 2 mM L-glutamine (VWR International, Radnor, PA, USA), 50 IU/mL penicillin, and 50 μg/ mL streptomycin (VWR International, Radnor, PA, USA) and incubated in a humidified atmosphere with 5% CO2 at 37 °C [24 (link)]. Cells were mostly cultured in 75 cm2 culture flasks and were collected using trypsin-EDTA (VWR International, Radnor, PA, USA) before the 80% of confluence was reached.
Trypsin edta
Manufactured by Avantor
Sourced in United States
Trypsin-EDTA is a solution used for cell detachment and dissociation in cell culture applications. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which work together to break down the extracellular matrix and cell-to-cell adhesions, allowing cells to be gently and efficiently removed from a culture surface.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Avantor (1 mentions)
Penicillin, by Avantor (1 mentions)
L glutamine, by Avantor (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
Roswell park memorial institute (rpmi), by Avantor (1 mentions)
Mcf 7, by Avantor (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Hepg2, by American Type Culture Collection (1 mentions)
Red blood cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Flow cytometry fixation buffer, by R&D Systems (1 mentions)
Hank s balanced salt solution hbss, by Avantor (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Dmem lg, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
Fitc phalloidin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
4 6 diamidino 2 phenylindole dapi staining solution, by Beyotime (1 mentions)
6 protocols using trypsin edta
1
Cell Line Cultivation and Maintenance
2
Cell Labeling and Flow Cytometry Protocol
3
Identification of Neoplastic Formation in Rat Bone Marrow MSC Transplantation
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Rat bone marrow mesenchymal stem cell (rBM-MSCs) were isolated from male Wistar rats. The cells in the femoral bone were flushed out with phosphate-buffered saline (PBS), collected by centrifugation at 800 g for 5 min, and cultured in Dulbecco's modified Eagle's medium with low glucose (LG-DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA). The medium was changed at 72 h after cells adhered to the culture dishes. When 80% confluence was reached, the cells were trypsinized with 0.25% trypsin-EDTA (Amresco, USA) and passed into new dishes for further expansion. Then the cells were identified as mesenchymal stem cells by surface marker identification. The male rat bone marrow MSCs (2×106) were intra-arterially injected into a female rat tail for experiments at day 0, 3, 7, 14, 21, we found that two of ten rats formed a foreign body in tails after 20 months and stripping out these bodies to process Hematoxylin–eosin (H&E) assay. Excitingly, it was considered as a novel neoplasm.
4
Bovine Uterine Epithelial Cell Isolation and Culture
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Initially, macroscopically healthy non-pregnant bovine uteri were carefully observed to be free of inflammation and abnormal color or any pathological lesions in slaughterhouse (Obihiro, Hokkaido, Japan), then collected and directly transferred to the laboratory under sterilized conditions and the uterine horn was used to isolate epithelial cells (2 (link), 7 (link)). The isolated cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12, Gibco, Grand Island, USA) supplemented with 1% amphotericin B, 0.1% gentamicin (Sigma-Aldrich, Steinheim, Germany), 10% heat-inactivated fetal calf serum (FCS) (Biowest USA) and 2.2% NaHCO3 using flask. The culture medium was replaced regularly with new media every 48 h. Upon reaching 70–80% confluence, the cells were collected with trypsinizing (0.05% trypsin EDTA; Amresco, Solon, OH, USA), transferred in 24-well and 12-well plates (Nalge Nunc International, Roskilde, Denmark) and cultured up to around 90% confluence (first passage). Estrogen (E2) and progesterone (P4) were added at preovulatory concentrations in the cell culture media (DMEM/F12, 1% amphotericin B, 0.1% gentamicin and 5% FCS) (2 (link), 7 (link)).
5
Mannitol and MTT Protocol for Cell Studies
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Mannitol (CAS:69–65-8) and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) kit were purchased from Aladdin (Shanghai, China), Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM-F12) was from Gibco (Invitrogen, Carlsbad, CA, USA), new-born calf serum (NCS) was from Tianhang (Hangzhou, China). The bicinchoninic acid (BCA) protein assay kit, 4',6-diamidino-2-phenylindole (DAPI) staining solution, malondialdehyde (MDA) assay kit were obtained from Beyotime Institute of Biotechnology, and glutathione (GSH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The Annexin V-FITC apoptosis detection kit was from Nanjing KeyGen Biotech Co. Ltd Nanjing, China. Trypsin-EDTA, Triton X-100, bovine serum albumin (BSA) were from Amresco (Solon, OH, USA). FITC-Phalloidin was obtained from Sigma (Chemical Co., St. Louis, MO, USA).
6
Isolation and Culture of hAF-MSCs
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The direct adherent method was used to separate hAF-MSCs [34 (link)]. In brief, hAF cells that were cultured in 25 cm2 flasks (Corning Incorporated, NY, USA) with expansion medium (BIOAMF-3TM Complete Medium) (Biological Industries, Kibbutz Beit Haemek, Israel) at 37 °C, 5% CO2 and 95% humidity were changed to culture with the basal growth medium comprised of Dulbecco's Modified Eagle Medium (DMEM)–high glucose (Gibco, USA) with a supplement of 10% fetal bovine serum (FBS) (Gibco, South America), gentamycin 40 mg/ml and Pen Strep (penicillin and streptomycin) 10,000 U/ml (Gibco, USA). The medium was changed every 3 days. After the cells reached 80% confluence, they were sub-cultured using 0.25% trypsin-EDTA (Gibco, USA). The cell samples that were collected from the 2nd passage were used in all of our experiments.
The hAF cell samples were observed under a DMi1 inverted phase contrast microscope (Leica Microsystems, USA). The cell samples that were collected from the 2nd passage were washed twice with sterile phosphate-buffered saline (PBS) (Amresco, Ohio, USA) and were trypsinized with 0.25% trypsin-EDTA. Subsequently, hAF cells were suspended in basal growth medium and centrifuged (C2 Series, Centurion Scientific Ltd, UK) at 2,035 g for 6 min at room temperature. After that, the supernatant was removed and the hAF cells were used in the experiments.
The hAF cell samples were observed under a DMi1 inverted phase contrast microscope (Leica Microsystems, USA). The cell samples that were collected from the 2nd passage were washed twice with sterile phosphate-buffered saline (PBS) (Amresco, Ohio, USA) and were trypsinized with 0.25% trypsin-EDTA. Subsequently, hAF cells were suspended in basal growth medium and centrifuged (C2 Series, Centurion Scientific Ltd, UK) at 2,035 g for 6 min at room temperature. After that, the supernatant was removed and the hAF cells were used in the experiments.
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