We obtained frozen aliquots of melanocytes isolated from foreskin of 106 healthy newborn males, mainly of European descent, following an established protocol (Halaban et al. 2000 (link)) from the SPORE in Skin Cancer Specimen Resource Core at Yale University. Cells were grown in lot-matched Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with lot-matched Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological) at 37°C with 5% CO2. Every step of cell culture, DNA/RNA isolation, and sequencing/genotyping processes was performed in rerandomized batches. Before harvesting the cells, media was taken and tested for mycoplasma contamination using MycoAlert PLUS mycoplasma detection kit (LT07-710, Lonza). All 106 samples were negative for mycoplasma contamination.
Amphotericin b penicillin streptomycin
Manufactured by Quality Biological
Amphotericin B/penicillin/streptomycin is a laboratory reagent that is commonly used as an antimicrobial supplement in cell culture media. It contains the antifungal agent Amphotericin B and the antibiotics penicillin and streptomycin, which work together to prevent microbial contamination in cell culture applications.
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2 protocols using amphotericin b penicillin streptomycin
1
Melanocyte isolation and culture protocol
2
Genotyping melanocyte cells for rs398206
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Melanocyte cells were grown in Dermal Cell Basal Medium (ATCC PCS-200-030) supplemented with Melanocyte Growth Kit (ATCC PCS-200-041) and 1% amphotericin B/penicillin/streptomycin (120-096-711, Quality Biological) as described before24 (link). Total RNA was isolated using a miRNeasy Mini kit (217004, Qiagen) further treated with CTAB-Urea following a previously described method68 (link) to remove excess melanin pigmentation. cDNA was synthesized from total RNA using iScript Advanced cDNA Synthesis Kit (Bio-Rad). Genomic DNA and cDNA were then genotyped for rs398206 using custom Taqman genotyping probe set (ANRWEYM) recognizing both genomic DNA and cDNA (ENST00000543692) with a 5 bp 5’ overhang on the left primer for cDNA based on Ensembl archive 75 annotation. From a total of 44 samples heterozygous for rs398206, 27 samples passing QC (Ct values lower than 38 for both alleles in cDNA and genomic DNA) were used to calculate A/C allelic ratio based on dRn values.
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