Cervical cancer derived cell line SiHa (HPV16 positive; ATCC® HTB-35TM, Manassas, VA, USA) and SW756 (HPV18 positive; ATCC® CRL-10302TM) were cultured in MEM medium supplemented with 10% fetal bovine serum (M10). Immortalized human embryonic kidney cells (HEK293T; ATCC® CRL-3216TM) were cultured in DMEM medium supplemented with 10% fetal bovine serum (D10). All cell culture procedures were performed at 37 °C and 5% CO2. For cell proliferation analysis cells were seeded in 24 well plates (1.0 × 104 cells/well) and cultured for 1 to 8 days. Cells were counted every 24 h using a hemocytometer.
Crl 3216
Manufactured by American Type Culture Collection
Sourced in United States, Germany, United Kingdom
The CRL-3216 is a laboratory equipment product from American Type Culture Collection. It is designed for the cultivation and maintenance of cell cultures. The core function of this product is to provide a controlled environment for the growth and propagation of various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (31 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (26 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (21 mentions)
Hek293t, by American Type Culture Collection (12 mentions)
Ccl 185, by American Type Culture Collection (9 mentions)
Ccl 2, by American Type Culture Collection (8 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (8 mentions)
Hb 8065, by American Type Culture Collection (6 mentions)
Htb 26, by American Type Culture Collection (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Tib 152, by American Type Culture Collection (5 mentions)
Opti mem, by Thermo Fisher Scientific (5 mentions)
Ccl 81, by American Type Culture Collection (5 mentions)
Hepes, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Tib 202, by American Type Culture Collection (4 mentions)
Htb 22, by American Type Culture Collection (4 mentions)
Htb 96, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Merck Group (4 mentions)
164 protocols using crl 3216
1
Cell Proliferation Analysis of Cancer Cell Lines
2
Cell culture of common cell lines
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Human embryonic kidney (HEK) cells from the 293T line (purchased from ATCC®, CRL-3216TM), human epithelial lung cells A549 (ATCC®, CCL-185TM), chinese hamster ovary (CHO) cells from the K1 line (ATCC®, CCL-61TM), human epithelial cervix cells HeLa (ATCC®, CCL-2TM) and human bone osteosarcoma epithelial cells U2OS (a kind gift from Ana García Sáez, University of Tübingen) were cultured in Dulbecco’s modified Eagle medium (DMEM) with the addition of fetal bovine serum (10%) and L-Glutamine (4 mM). Cells were passaged every 3–5 days, no more than 15 times. All solutions, buffers and media used for cell culture were purchased from PAN-Biotech (Aidenbach, Germany).
3
Imaging of pH-sensitive fluorescent proteins
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Human embryonic kidney (HEK) cells from the 293T line (purchased from ATCC®, Manassas, VA, USA, CRL-3216TM) and human epithelial lung cells A549 (ATCC®, CCL-185TM) were cultured in Dulbecco's modified Eagle medium (DMEM) with the addition of fetal bovine serum (10%) and L-Glutamine (2 mM). Cells were passaged every 3-5 days, no more than 15 times. All solutions, buffers and media used for cell culture were purchased from PAN-Biotech (Aidenbach, Germany).
For microscopy experiments, 3 × 10 5 (HEK) or 4 × 10 5 (A549) cells were seeded in 35 mm #1.5 optical glass bottom dishes (CellVis, Mountain View, CA, USA) 24 h before transfection.
Cells were transfected 16-24 h prior to the experiment using between 50 ng and 150 ng plasmid per dish with Turbofect (HEK) or Lipofectamin3000 (A549) according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, plasmids were incubated for 20 min with 3 µl Turbofect diluted in 50 µl serum-free medium, or 15 min with 2 µl P3000 and 2 µl Lipofectamine3000 diluted in 100 µl serum-free medium, and then added dropwise to the cells. For spectral imaging at different pH values, culture medium was exchanged with buffer containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES with pH ranging from 5.0 to 9.2.
For microscopy experiments, 3 × 10 5 (HEK) or 4 × 10 5 (A549) cells were seeded in 35 mm #1.5 optical glass bottom dishes (CellVis, Mountain View, CA, USA) 24 h before transfection.
Cells were transfected 16-24 h prior to the experiment using between 50 ng and 150 ng plasmid per dish with Turbofect (HEK) or Lipofectamin3000 (A549) according to the manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, plasmids were incubated for 20 min with 3 µl Turbofect diluted in 50 µl serum-free medium, or 15 min with 2 µl P3000 and 2 µl Lipofectamine3000 diluted in 100 µl serum-free medium, and then added dropwise to the cells. For spectral imaging at different pH values, culture medium was exchanged with buffer containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES with pH ranging from 5.0 to 9.2.
4
Culturing Mouse and Human Macrophages
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Mouse iBMDMs were gifts from Dr. Jonathan Kagan. Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Corning 15013CV) supplemented with 10% fetal bovine serum (FBS, Corning 35–016-CV), 2 mM L-glutamine (Corning, MT25005CI), and 0.1% penicillin/streptomycin (P/S, Corning, MT3000CI) at 37°C in humidified incubators with 5% CO2. Cells were passaged every 2–3 days when they reached 60%–80% confluency. Human embryonic kidney 293T (HEK293T, ATCC, CRL3216) cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 0.1% penicillin/streptomycin. Human THP1 macrophages (ATCC, TIB202) were cultured in RPMI 1640 (ATCC modification, Gibco A1049101) supplemented with 10% FBS and differentiated with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, P8139) for 24 hours. Cell lysate of primary mouse bone marrow-derived macrophages was a gift from the laboratory of Dr. Yuan He at Wayne State University (66 (link)).
5
Porcine Cell Line Maintenance and PCV2 Propagation
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The PK-15 cell line (CCL-33, ATCC, Manassas, VA, USA) was maintained in minimal essential medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Human embryonic kidney epithelial (HEK) 293T cells (CRL-3216, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco). The DDX21-knockdown PK-15 cells were constructed as previously described (Zhou et al., 2022a (link)). All media were supplemented with 10% fetal bovine serum (S711-001S; LONSERA, Shanghai Shuangru Biology Science & Technology Co., Ltd, China) as previously described (Zhou et al., 2022c (link)). PCV2 strain BJW (accession no. AY847748.1 ) was propagated in PK-15 cells (Liu et al., 2005 (link)).
6
Dorsal Spinal Cord Explant Guidance
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Dorsal spinal cord explants form E12.5 mouse embryos were dissected and cultured in collagen gels as previously described (Serafini 1994). Briefly, explants were embedded in rat tail collagen (Corning, #354249) gels at a distance of one explant diameter away from a mock 293 T cell aggregate (ATCC, CRL-3216) or a cell aggregate expressing Slit (pSecTagB-hSlit2-MYC, kind gift from A. Chedotal). Explants were grown in 50% OptiMEM and 45% Ham’s F-12 media supplemented with 5% HS, 0.75% glucose, and 1X penicillin/streptomycin/glutamine for 48 hr with 500 ng/ml Netrin-1. Explants were subsequently fixed and stained as described above. For preparation of 293 T cell aggregates, cells were trypsinized and resuspended in a rat tail collagen solution, drawn into a glass Pasteur pipette, and allowed to polymerize. The collagen-embedded cells were released from the pipette using a rubber bulb and the aggregates cut into 1 mm clusters.
7
Diverse Cell Lines for Research
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Cell lines used in this study were obtained from ATCC: Human cells epithelial embryo (HEK293T, CRL-3216), Felis catus kidney cells (CRFK, CCL-94), Canis familiaris epithelial kidney cells (MDCK, CCL-34), Canis familiaris tumor fibroblast cells (A-72, CRL-1542) and Sus scrofa pig testis fibroblast cells (ST, CRL-1746) or ThermoFisher Scientific: ExpiCHO cells and Expi293F™ cells. Cells were cultivated at 37°C, in an atmosphere of 5 % CO2 and with 130 rpm of agitation for suspension cells. None of the cell lines used were routinely tested for mycoplasma contamination.
8
Lentiviral Delivery of miR-301b Mimics
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Lentivirus containing pre-miRNA-301b expressed in a lentiviral vector (pLKO.1-puro) were generated in 293 T cells as previously described [33 (link)]. Briefly, the coding oligo-nucleotides of antisense human miR-301b mimics and NC sequence were cloned and inserted into a lentivirus expression vector, pLKO.1-puro. The miR-301b mimics and NC viral particles were produced in 293 T cells (CRL-3216; ATCC, Manassas, VA, USA) via lentivirus expression vector co-expressed with pPACK packaging system (Systems Biosciences, Palo Alto, CA, USA). A375 and A2058 cells were transfected by miRNA 301b-GFP-expressing lentiviruses with the multiplicity of infection or MOI equals to 40. Transfection monitoring was performed by observing the GFP positive cells under a Fluorescent microscopy.
9
Efficient Lentiviral Transduction of MSCs
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Viral particles were produced in human embryonic kidney 293T cells (ATCC® CRL-3216™ www.atcc.org accessed on 22 March 2021). Briefly, 293T cells were seeded in high-glucose DMEM containing 10% FBS. pMD2.G (Addgene, 12259), psPAX2 (Addgene, 12260) and the lentiviral vector carrying the transgene were transfected into the packaging cell line by calcium phosphate precipitation. MSC transduction was carried out at a multiplicity of infection of 10 in order to achieve >95% infection. pLV-hTERT-IRES-hygro was obtained from Addgene (Addgene, 85140). The efficiency of transduction was evaluated by a hygromycin selection test. The pWPI-HIF-1α-GFP and pWPI-GFP vectors have previously been described [18 (link)]. Transduction efficiency was evaluated by quantifying GFP+ cells by flow cytometry. MSC-T cells were transduced with pWPI-GFP as a control (named MSC-T). GFP-positive cells were sorted to obtain pure cultures. Population doubling times (PDT) were calculated using the formula PDT = (t2 − t1)/3.32 × (logN2 − logN1) where N2 is the cell harvest number and N1 the inoculum cell number [46 (link)].
10
Cell Culture Conditions for Common Cell Lines
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All cells were incubated at 37° C and 5% CO2 and routinely checked for mycoplasma contamination. HeLa cells, which are derived from cervix adenocarcinoma (CCL-2, American Tissue Cell Culture (ATCC)) were cultured in EMEM (SH3024401, Hyclone) supplemented with 10% FBS. 293T cells, which are human embryonic kidney (HEK) cells that express and are transformed with large T antigen (CRL-3216, ATCC) were cultured in DMEM (25–501N, Genesee) with 10% FBS. Tera-1 cells, which originate from metastatic human GCT/teratoma (HTB-105, ATCC) were cultured in McCoy’s 5A (16600–082, Life Technologies) supplemented with 20% cosmic serum (SH3008702, Fisher Scientific).
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