Human embryonic kidney cells (HEK-293T) and HeLa cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Thermo Fisher Scientific), 100 U/l penicillin G sodium, and 100 µg/ml streptomycin sulfate. Cells were kept under 95% humidified air and 5% CO2 at 37°C. For immunofluorescence, cells were seeded on glass coverslips before transient transfection with Lipofectamin 3000 (Thermo Fisher Scientific) following the manufacturer’s instructions. Protein expression for protein biochemistry was performed by treating 80% confluent T75 tissue culture flasks (Sarstedt, Nümbrecht, Germany) with a mixture of 635 µl ddH2O, 815 µl 300 mM NaCl, 180 µl polyethylenimine solution (0.323 g/l; Polysciences, Warrington, PA), and 22 µg of plasmid DNA. The O-glycosylation blocker benzyl-2-acetamido-2-deoxy-α-d -galactopyranoside (BenGal) and the N-glycosylation inhibitor tunicamycin (Sigma Aldrich, St. Louis, MO) were dissolved in DMSO to stock solutions of 1 mg/ml and 300 mM, respectively, and stored at –20°C. Indicated cell culture media were supplemented with 1 µg/ml tunicamycin, 300 µM BenGal, or equal amounts of DMSO.
T75 tissue culture flasks
Manufactured by Sarstedt
Sourced in Germany, Canada
The T75 tissue culture flask is a laboratory equipment used for cell culture. It provides a controlled environment for the growth and maintenance of cells. The flask has a surface area of approximately 75 cm2, which allows for the cultivation of a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
U shaped 96 well plates, by Greiner (2 mentions)
96 well tissue culture plate, by Sarstedt (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Lipofectamin 3000, by Thermo Fisher Scientific (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Murine llc cells, by American Type Culture Collection (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Ventilation cap, by Sarstedt (1 mentions)
Rpmi 1640 medium, by Biosera (1 mentions)
Penicillin streptomycin, by Biosera (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Porcine gelatin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
15 protocols using t75 tissue culture flasks
1
Cell Culture and Glycosylation Inhibition Protocols
2
Mouse Cell Line Culturing Protocol
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Mouse L929 fibroblasts, 4T1 murine mammary carcinomas, and murine LLC cells were obtained from ATCC (Manassas, VA, USA). E0771 murine mammary carcinoma cells[34 (link)], ID8 ovarian carcinoma cells[35 (link)], and HC11 mouse mammary epithelial cells [36 (link)] were kind gifts from Drs. David Waisman, Craig McCormick, and Hyo Sung Ro (all from Dalhousie University, NS, Canada) respectively. All cell lines were grown in T75 tissue culture flasks (Sarstedt, QC, Canada) and maintained at 37°C and 5% CO2 in DMEM containing 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 5 mM HEPES (ph 7.4), hereafter referred to as “DMEM” unless otherwise indicated. At 90% confluence, cells were passaged by detachment with trypsin-EDTA, followed by centrifugation at 500 x g for 5 and then resuspension in fresh DMEM. Freshly isolated BMDMs were cultured at 37°C at 5% CO2 in RPMI-1640 medium containing 10% FCS (v/v), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 5 mM HEPES (ph 7.4), hereafter referred to as “RPMI-1640” unless otherwise indicated. Prior to experimental use, BMDMs were detached with EDTA (10 mM in PBS), centrifuged at 500 x g for 5 min, and resuspended in fresh RPMI-1640 before cell seeding.
3
Generating B-cell Cultures Using Irradiated 40LB Fibroblasts
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The iGB culture system was described previously by Nojima et al.38 (link). Briefly, the 3T3 fibroblast cell line of BALB/c origin stably expressing CD40 ligand and B cell activating factor (BAFF) (40LB cell line), was cultured and maintained in high-glucose DMEM with GlutaMAX (catalog no. 31966021, Thermo Fisher Scientific) medium supplemented with 10% FCS and 50 U ml−1 penicillin/streptomycin (catalog no. 15070063, Thermo Fisher Scientific) in T75 tissue culture flasks (catalog no. 658175, Sarstedt). Once cells were confluent, they were detached using trypsin/EDTA (catalog no. 25200056, Gibco) treatment, washed and collected in 15-ml tubes in 5 ml medium and irradiated (80 Gy). After irradiation, cells were washed, counted and seeded at 3 × 106 per dish (100 mm, catalog no. G664160, Greiner) or 0.5 × 106 per well (6-well plate, catalog no. 353046, Falcon). Fibroblast attachment and stretching were allowed overnight at 37 °C and 5% CO2. The next day, naive B cells were isolated using anti-CD43 microbeads and treated with TAT-Cre (approximately 1.5 μM or 66.7 U ml−1, catalog no. SCR508, Merck) for 45 min as described in Supplementary Methods . After three washes, cells were counted and cultured on an irradiated 40LB layer at 5 × 105 (100-mm dish) and 5 × 104 (per well, 6-well plate) for 4–6 days.
4
CT-26 Cell Culturing Protocol
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CT-26 cells were obtained from ATCC and cultured in RPMI-1640 Medium (BioSera, Boussens, France), supplemented with 10% fetal bovine serum (BioSera, Boussens, France) and 1% Penicillin/Streptomycin (BioSera, Boussens, France) in sterile T75 tissue culture flasks with ventilation caps (Sarstedt, Nümbrecht, Germany) in a humidified 5% CO2 atmosphere at 37 °C. Cells were cultured for a maximum of 25 passages or 60 days after thawing and were screened for mycoplasma infection.
5
Transient Transfection of HEK-293T and HeLa Cells
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Human embryonic kidney (HEK-293T) cells and HeLa cells (ATCC, Wesel, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate in an atmosphere of 95% humidified air and 5% CO2 at 37 °C. Transient transfections were performed by incubating 80% confluent cells in T75 tissue culture flasks (Sarstedt, Nümbrecht, Germany) overnight with 815 µL of 300 mM NaCl, 635 µL of ddH2O, 180 µL of polyethylenimine (0.323 g/L, Polysciences, Warrington, PA, USA) and 22 µg of plasmid DNA. For immunofluorescence staining, HeLa cells were seeded on glass coverslips prior to performing transient transfection with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. When indicated, the cell culture media were supplemented with 1 µg/mL tunicamycin, 300 µM BenGal or an equal amount of the respective solvent (DMSO).
6
Hypoxia-induced endothelial-smooth muscle cell co-culture
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HPAECs (PromoCell, Cat. no. C-12241) from 3 to 4 different biological donors were cultured in T75 tissue culture flasks (Sarstedt, Germany, Cat. no. 833911002) coated with 10 μg/mL Fibronectin (10 μg/mL, EMD Millipore Corp, USA; 341631) in endothelial cell growth medium 2 (ECGM-2, Promocell, Germany C-22211), supplemented with 2% foetal calf serum (FCS) and growth factor mix (PromoCell, Cat. no. C-22111), 1% Streptomycin/Penicillin (100 μg/mL) and 1% MycoZap™.
HPASMCs (Lonza, Cat. No. CC-2581) were cultured in T75 tissue culture flasks coated with 0.2% porcine gelatin (Sigma, Cat. no. G1890) in smooth muscle cell growth medium 2 (SmGM-2, PromoCell, Cat. no. C-22062), supplemented with 5% FBS and growth factor supplement (PromoCell, Cat. no. C-39267) and antibiotics. Cells were acquired at passage 3 and were used for experiments in passages 4–6. Donor information is provided in Supplementary Table2 . For HPAEC and HPASMC co-culture, ECGM-2 medium was supplemented with 10% foetal bovine serum (FBS). VEGF, ascorbic acid, heparin, and hydrocortisone, known to inhibit HPASMC proliferation, were excluded.
To induce hypoxia, cells were incubated in a humidified 37 °C incubator set at 5% CO2 and 2% O2.
HPASMCs (Lonza, Cat. No. CC-2581) were cultured in T75 tissue culture flasks coated with 0.2% porcine gelatin (Sigma, Cat. no. G1890) in smooth muscle cell growth medium 2 (SmGM-2, PromoCell, Cat. no. C-22062), supplemented with 5% FBS and growth factor supplement (PromoCell, Cat. no. C-39267) and antibiotics. Cells were acquired at passage 3 and were used for experiments in passages 4–6. Donor information is provided in Supplementary Table
To induce hypoxia, cells were incubated in a humidified 37 °C incubator set at 5% CO2 and 2% O2.
7
HEK293 Cell Transfection Protocol
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HEK293 cells were cultured in DMEM (41965039, Gibco) supplemented with 10% fetal bovine serum (FBS; 16250078, Life Technologies) and 1% penicillin-streptomycin (15140122, Life Technologies) in T75 tissue culture flasks (83.3911.002, Sarstedt) at 37°C in a humified atmosphere and 5% CO2. 60% confluent cells were transiently transfected with DNases coding pcDNA3.1 vectors using polyethyleneimine (PEI; 23966-2, Polysciences). After 48h transfection supernatants were removed and centrifuged for 5 min at 500 x g.
8
Maintaining and Characterizing Cancer Cell Lines
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Cell lines were acquired from the American Tissue Culture Collection (ATCC) (LGC Standards, Middlesex, UK). Human prostate carcinoma cells, DU-145 (ATCC-HTB-81), human lung adenocarcinoma cells, A-549 (ATCC-CCL-185) and human mammary epithelial adenocarcinoma cells, MCF-7 (ATCC-HTB-22) were maintained in basic Dulbecco’s modified Eagle’s medium (Gibco-BRL, Paisley, UK) supplemented with 10 % foetal calf serum (FCS) and 1 % (vol/vol) Penicillin/Streptomycin (both from Sigma, Ireland) (complete medium). Depending on cell type, supplements were added according to ATCC instructions. Cells were maintained in 5 % CO2 in a humidified atmosphere at 37 °C and were routinely seeded into T75 tissue culture flasks (Sarstedt, Ireland) and grown to a maximum of 80–90 % confluence. At seeding and prior to experiments, cells were washed in Phosphate Buffered Saline pH 7.4 (PBS) to remove cellular debris and dead cells. Cells were detached using 0.5 % Trypsin-EDTA (Sigma-Aldrich) for 5 min, counted on the Countess Cell Counter (Life Technologies, Paisley, UK). Passage numbers did not exceed 25. For all experiments (except untreated cells), cells were supplemented with 200 μm dimethyloxaloglycine (DMOG).
9
Isolation of Rabbit PBMCs Using OptiPrep
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Fresh blood collected from female New Zealand white rabbits (Covance Research Products, Denver, PA, USA) was used in this study. Peripheral blood mononuclear cells (PBMCs) including monocytes were isolated using OptiPrep (Axis‐Shield, Oslo, Norway) according to a modified manufacturer's protocol for rabbit blood. Twofold diluted rabbit's blood was layered on OptiPrep density solution. After density gradient centrifugation at 700 g for 20 min with nonaccelerator and nonbrake modes, isolated PBMCs were washed two times with phosphate‐buffered saline (PBS; GE Healthcare Life Sciences Hyclone Laboratories, Logan, UT, USA) by centrifugation at 250 g for 10 min. Cells were resuspended in RPMI‐1640 medium (Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences Hyclone Laboratories) and 1% penicillin/streptomycin (Life Technologies) at a concentration of 2 × 106 cells·mL−1. Subsequently, cell suspensions were transferred to T75 tissue culture flasks (Sarstedt, Newton, NC, USA), four‐well chamber slides (Thermo Fisher Scientific, Rochester, NY, USA), or 24‐well flat bottom tissue culture plates (Becton Dickinson, Flanklin Lakes, NJ, USA). After incubation for 2 h at 37 °C in humidified 5% CO2 in a gas incubator (Galaxy 170R; Eppendorf, Enfield, CT, USA), nonadherent cells were removed by washing with PBS 15 .
10
Cultivation of Various Cell Lines
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4T1 murine mammary carcinoma cells (CRL-2539), CT26 murine colon carcinoma cells (CRL-2638), U2OS human osteosarcoma cells (HTB-96), and NIH/3T3 normal murine fibroblast cells (CRL-1658) were purchased from the American Type Culture Collection (Manassas, VA). MCA205 murine fibrosarcoma cells (SCC173) and DC2.4 murine dendritic cells (SCC142) were purchased from MilliporeSigma (Burlington, MA). Cell lines were mycoplasma-tested before being used. 4T1 cells, MCA205 cells, and DC2.4 cells were propagated in RPMI 1640 (Corning, Corning, NY) supplemented with 10% FBS (MilliporeSigma). NIH/3T3 cells and U2OS cells were propagated in DMEM (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. HUVECs were propagated in the EGM-2 bulletkit (LONZA, Basel, Switzerland). All cell lines were grown in T75 tissue culture flasks (Sarstedt, Nümbrecht, Germany) under standard culture conditions of 37 °C and 5% CO2.
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