Immortalized human fibroblasts derived from a FA complementation group P patient with bi-allelic SLX4 mutations or derivates complemented with wild-type protein or dissociation-of-functions mutants were a kind gift from Dr. A Smogorzewska, Rockefeller University, New York, NY [11 (link)]. These mutants were deleted (Δ) for either one of three interaction domains: ΔXPF (MLR), ΔMUS81 (SAP), or ΔSLX1 (SBD). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) with 1 μg/ml puromycin and 20% Bovine Growth Serum (BGS) (HyClone, South Logan, Utah). Immortalized mouse embryonic fibroblasts (MEF) derived from Mus81 wild-type (+/+) or deficient (−/−) mice were kindly provided Drs. R. Hakem and P. McPherson, University of Toronto, Toronto, Canada [21 (link)]. Cells were maintained in DMEM with 1 μg/ml puromycin. Chinese hamster ovary (CHO) cell lines AA8 and the ERCC1-mutant line UV20 were cultured in modified McCoy's 5a medium (Gibco, Carlsbad, CA). All cell lines were maintained in a humidified incubator at 37°C and 5% CO2, with 10% BGS, 20 mM HEPES, 1% Streptomycin-Penicillin and 2 mM L-Glutamine (Sigma-Aldrich, St. Louis, MO) supplemented to the medium. Cells were tested mycoplasma free (MycoAlert, Lonza, Walkersviller, MD) and used within 10 passages, with 2-3 passages per week, for all experiments after resuscitation.
Bovine growth serum (bgs)
Manufactured by Merck Group
Sourced in United States
The BGS is a lab equipment product manufactured by Merck Group. It is a device used for the separation and analysis of biological samples. The core function of the BGS is to perform size-based fractionation of macromolecules, such as proteins and nucleic acids, through the application of a gel-based separation technique.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Mycoalert, by Lonza (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Oxytocin, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
4 protocols using bovine growth serum (bgs)
1
Immortalized Fibroblasts for SLX4 Mutations
2
Evaluating T Cell Proliferation with Myeloid Cells
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The MoDCs or monocytes were cultured in triplicate in U-bottom 96-well culture plates and mixed with 1.1 x 105 allogenic T cells/well containing media alone or media supplemented with either BGs (40 particles/cell), ConA (0.5 ng/well) or LPS (100 ng/well) from E. coli (Sigma Aldrich, USA). For this purpose, T cells were purified from PBMCs over a nylon wool column as described elsewhere [25 ]. The proliferation of the T cells was measured using MTT based assay as described earlier [26 (link)]. The T cell proliferative responses for each culture condition were expressed as a stimulation index (SI), where SI was calculated by measuring absorbance of T cells and myeloid cells divided by absorbance of T cells in a medium.
3
Cardiac Progenitor Sphere Generation
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For generation of CardioStem spheres,21 0.25 to 0.5×105 mCPCs were seeded on 6‐well dishes with culture medium (35% Iscove's modified Dulbecco's medium, 32.5% DMEM, 32.5% F12, and 3.5% bovine growth serum [HyClone]) with 100 nmol/L oxytocin (Sigma) for 3 days, then trypsinized and cultured on bacterial dishes (P100) for 2 to 3 days to induce CardioStem sphere formation. For rCPCs, 1.0×105 cells were seeded on 6‐well dishes with culture medium (F12 with 10% FCS) with 100 nmol/L oxytocin for 3 days, then trypsinized and cultured on bacterial dishes (P100) for 3 days.
4
SH-SY5Y Cells for Drug Screening
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SH-SY5Y-GLuc-SERCaMP cells were kindly provided by Dr. Brandon Harvey of the National Institute on Drug Abuse i.r.p., NIH. Cells were cultured in a 37 °C humidified incubator with 5% CO2 in DMEM (4.5 g/L D-GLucose) containing 2 mM GlutaMAX, 10% bovine growth serum (Sigma Aldrich), 10 U/mL penicillin (Thermo Fisher Scientific), and 10 μg/mL streptomycin (Thermo Fisher Scientific). Cells were plated at 5 × 104 cells per well (100 μL volume). Media were exchanged into DMEM (4.5 g/L D-GLucose) containing 2 mM GlutaMAX, 1.5% BGS, 10 U/mL penicillin and 10 μg/mL streptomycin before 16-h drug pre-treatment. Cells were incubated for 48 h prior to adding drugs. Media was collected (5 μL) prior to and at indicated time points post-drug treatment for enzymatic assay.
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