The human colon cancer cell line HCT116 was acquired from ATCC and served as an in vitro model for cancerous intestinal cells. HCT116 cultures were performed according to the specification of the supplier using McCoys 5A medium (Gibco REF 22330-021) supplemented with 10% (v/v) fetal calf serum (FCS) 1% (v/v) Penicillin/Streptomycin in TC-Flasks (Sarstedt). The immortalized human colonic epithelial cell line HCEC-1CT was kindly provided by Prof. Jerry W. Shay (UT Southwestern Medical Center, Dallas, TX, USA). HCEC-1CT cultures were performed according to previously described protocol (Rebhahn et al. 2022 (link); Warth et al. 2016 (link)) using Dulbecco’s Modified Eagle Medium 92.8% (Gibco, REF 21063-029), Medium 199 (10x) 2%, Cosmic Calf Serum 2%, 1 M HEPES buffer solution 2%, Insulin–Transferrin–Selenium [100 µg/ml] 1.04%, Gentamicin Solution 0.12%, Epidermal Growth Factor [100 µg/ml] 0.02%, and Hydrocortisone [5 mg/ml] 0.02%. For cultivation and incubations, humidified incubators were used at 37 °C and 5% CO2. For assays, cells were grown to 80–90% confluency.
Penicillin streptomycin
Manufactured by Sarstedt
Sourced in Germany
Penicillin/Streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of the antibiotics penicillin and streptomycin, which work together to inhibit the growth of a broad spectrum of bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Tc flasks, by Sarstedt (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cd11b microbead isolation kit, by Miltenyi Biotec (1 mentions)
Dmem glutamax, by Sarstedt (1 mentions)
Poly l lysine (pll), by Provitro (1 mentions)
Fetal bovine serum (fbs), by Sarstedt (1 mentions)
Dmem glutamax, by Thermo Fisher Scientific (1 mentions)
T75 flask, by Sarstedt (1 mentions)
Fibroblast growth kit low serum, by American Type Culture Collection (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Collagenase, by Roche (1 mentions)
5 protocols using penicillin streptomycin
1
In Vitro Colon Cancer and Epithelial Cell Culture
2
Isolation of Postnatal Mouse Microglia
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Mice aged P0–P3 were euthanized by decapitation, and brains were dissected into PBS on ice. Brains of 6–8 mice were pooled, centrifuged at 500 × g for 10 min at 4 °C and resuspended in 0.25% Trypsin-EDTA (Thermo Fisher Scientific) at 37 °C for 10 min. DNase I (Thermo Fisher Scientific) was added at 1 mg/ml to the solution, and the brains were digested for 10 more minutes at 37 °C. Trypsin was neutralized by adding DMEM + GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cells were passed through a 70 µm cell strainer. Cells were centrifuged at 500 × g for 10 min at 4 C, resuspended in DMEM + GlutaMAX with 10% FBS 1% penicillin/streptomycin and cultured in T-75 flasks (Sarstedt), pre-coated with 2 µg/ml Poly-L Lysine (PLL, Provitro) at 37 °C in a humidified incubator with 5% CO2 for 5–7 days until confluence was reached. Mixed glial cells were shaken for 30 min at 180 rpm, the supernatant was collected and the medium was changed, and then cells were shaken for at least 2 h at 220 rpm and the supernatant was collected and the medium was changed again. CD11b+ microglia were isolated from the collected supernatant using the CD11b Microbead Isolation kit (Miltenyi) according to the manufacturer’s instruction and seeded onto PLL-coated plates.
3
Cultivation of Bladder Cancer and Fibroblast Cells
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The urinary bladder carcinoma cell line T24 (ATCC® HTB4™ USA) was purchased from the American type culture collection (ATCC; Virginia, USA). Cells were cultivated according to the specification of the supplier in McCoy´s 5a medium (Gibco, Netherlands) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% (v/v) Penicillin/Streptomycin in TC-Flasks T25/T75 (Sarstedt, Nümbrecht, Germany). Normal, human, primary bladder fibroblasts (PCS-420–013™) were purchased from the American type culture collection (ATCC; Virginia, USA). Cells were cultivated in Fibroblast Basal Medium (ATCC, PCS-201–030) supplemented with Fibroblast Growth Kit-Low Serum (ATCC PCS-201–041), containing final concentrations of 5ng/ml rhFGF, 7.5mM L-glutamine, 50µg/ml ascorbic acid, 1µg/ml hydrocortisone hemisuccinate, 5µg/ml rh insulin, and 2% FBS. For cultivation and incubations, humidified incubators were used at 37 °C and 5% CO2 if not otherwise specified. Cells were regularly passaged when reaching a confluency of 80 – 90% (3 times a week).
4
Isolation and Culture of Human Mesenchymal Stem Cells
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Human MSC isolation was performed after written consent from the bone marrow of healthy donors (n = 5). The production and use of hMSCs has been approved by the Ethics Committee of Hannover Medical School, Germany (approval no. 2858–2015).
The puncture was performed after local anesthesia with a bone marrow puncture needle 15 G (Almond & Rupp, Erkrath, Germany) at the pelvic crest. Approximately 10–15 mL of bone marrow was aspirated. Human bone marrow was diluted with a ratio of 1:4 with sterile phosphate-buffered saline (PBS) and the cell count was determined. Cells were cultivated with a concentration of 1–2 × 106 cells/cm2 with 50 mL CellGro® MSC medium (Cellgenix, Freiburg im Bresgau, Germany) and enriched with 1% Penicillin/Streptomycin, heparin 2 IU/mL, and 8% human plate lysate in 175 cm2 tissue culture flasks (Sarstedt, Nümbrecht, Germany). After four days, cells were washed thrice with 30 mL PBS to avoid blood residues. The medium was changed every 4–5 days and adherent cell growth was microscopically controlled. Cells were subcultured until they reached 80% of confluence and passaged at a density of 2 × 103 cells/cm2. The remaining cells were cryopreserved at a density of 1 × 106 cells/mL in MSC medium with 5% dimethylsulfoxide (DMSO, Sigma Aldrich, Taufkirchen, Germany) and frozen at 70 °C. Cells were used for culture at a low passage (≤4) [23 (link),24 (link)].
The puncture was performed after local anesthesia with a bone marrow puncture needle 15 G (Almond & Rupp, Erkrath, Germany) at the pelvic crest. Approximately 10–15 mL of bone marrow was aspirated. Human bone marrow was diluted with a ratio of 1:4 with sterile phosphate-buffered saline (PBS) and the cell count was determined. Cells were cultivated with a concentration of 1–2 × 106 cells/cm2 with 50 mL CellGro® MSC medium (Cellgenix, Freiburg im Bresgau, Germany) and enriched with 1% Penicillin/Streptomycin, heparin 2 IU/mL, and 8% human plate lysate in 175 cm2 tissue culture flasks (Sarstedt, Nümbrecht, Germany). After four days, cells were washed thrice with 30 mL PBS to avoid blood residues. The medium was changed every 4–5 days and adherent cell growth was microscopically controlled. Cells were subcultured until they reached 80% of confluence and passaged at a density of 2 × 103 cells/cm2. The remaining cells were cryopreserved at a density of 1 × 106 cells/mL in MSC medium with 5% dimethylsulfoxide (DMSO, Sigma Aldrich, Taufkirchen, Germany) and frozen at 70 °C. Cells were used for culture at a low passage (≤4) [23 (link),24 (link)].
5
Isolation and culture of thyrocytes
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Surgical thyroid tissue was obtained from 3 patients with HT and 3 benign nodular thyroid, euthyroid at the time of surgery. Thyroidectomy was adviced to these patients mainly because of the presence of a large goiter and/or thyroid nodules. The study has been conducted along the lines of the Declaration of Helsinki (2000) on the ethic in clinical study. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and national laws; the patients gave their informed consent to it [50] .
Thyrocytes were prepared as reported previously [51, 52] . Surgical tissues were minced with scissors and digested with collagenase (1 mg/mL; Roche, Mannheim, Germany) in RPMI 1640 (Gibco BRL, Paisley, UK) for 1 h at 37 °C. Semi-digested follicles were removed, sedimented for 2 min, washed, and cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM glutamine, and 50 µg/mL penicillin/streptomycin at 37 °C and 5% CO 2 in plastic 75 cm 2 flasks (Sarstedt, Verona, Italy).
Thyrocytes were prepared as reported previously [51, 52] . Surgical tissues were minced with scissors and digested with collagenase (1 mg/mL; Roche, Mannheim, Germany) in RPMI 1640 (Gibco BRL, Paisley, UK) for 1 h at 37 °C. Semi-digested follicles were removed, sedimented for 2 min, washed, and cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM glutamine, and 50 µg/mL penicillin/streptomycin at 37 °C and 5% CO 2 in plastic 75 cm 2 flasks (Sarstedt, Verona, Italy).
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