Skin punch biopsies were obtained from an affected individual homozygous for RRP7A c.465 G > C and from a MCPH patient homozygous for a WDR62 mutation. The samples were treated with Dispase II (1.5 U ml−1, Roche) in phosphate-buffered saline (PBS) at 4°C for 3 h to separate epidermis from dermis. The dermis was then incubated at 37°C and 5% CO2 in DMEM (Invitrogen) supplemented with 10% foetal bovine seuym (FBS, Sigma; F9665), 1% GlutaMAXTM (GIBCO®), 100 IU ml−1 penicillin (pen), and 100 μg ml−1 streptomycin (strep) (Sigma; P0781). Culture medium was changed after every 3−4 days and a pool of human dermal fibroblasts (HDFs) was expanded by further sub-culturing. Similarly, control HDFs were obtained using skin biopsy from 34 years old healthy control female of Asian origin. hTERT RPE-1 (RPE-1; ATCC, CRL-4000) cells were cultured in DMEM medium supplemented with 10% FBS and 5% pen/strep. HEK293T cells (ATCC, CRL-3216) were cultured in DMEM containing high l -glucose concentration (GIBCO®) supplemented with 10% FBS and 1% pen/strep. Culture medium was changed every 2–3 days for both RPE-1 and HEK293T cells.
Streptomycin strep
Manufactured by Merck Group
Sourced in United States
Streptomycin is an antibiotic medication used to treat various bacterial infections. It is produced by the bacterium Streptomyces griseus. Streptomycin functions by inhibiting protein synthesis in bacteria, which leads to their death or inhibition of growth.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Dispase 2, by Roche (1 mentions)
Pen strep, by American Type Culture Collection (1 mentions)
2 protocols using streptomycin strep
1
Skin Biopsy for Fibroblast Isolation
2
Cement Elution Protocol for Cell Culture
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Cement elutes were prepared as previously described29 . In short, cements were mixed inside a laminar flow hood and inserted into 1000 µl pipette tips (VWR, Radnor, PA, USA) that were cut at 2.2 cm from their final segment. The tip with the cement was attached to the lid of a microcentrifuge tube using an o-ring (5 mm in diameter and 2 mm in height). Upon closing of the lid, the tip containing the cement was immersed in the tube containing 0.5 ml of Dulbecco’s Modified Eagle Medium (DMEM) (ATCC, Manassas, VA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Gibco, Grand Island, NY), 100 U/mL penicillin (PEN) and 100 mg/mL streptomycin (STREP) (Sigma-Aldrich, Saint Louis, MO, USA). Each cement sample was incubated at 37°C, 95% humidity and 5% CO2 for 24 and 48 hours. After each incubation period, the samples were removed from the media and the extract media were briefly vortexed, transferred to a new microcentrifuge tube and stored at -20oC until further use.
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