Klapperich Lab:Primary Neonatal Foreskin Fibroblasts
Introductory procedure on growing primary neonatal foreskin fibroblasts.
Human neonatal foreskin from circumcisions performed at Brigham and Women's hospital are obtained. The foreskins are washed exhaustively in PBS and then may be stored in media at 4 C for up to 24 hours after excision. The epidermal and dermal layers are pulled apart using forceps and scalpel and the epidermis is reserved for other applications. The dermal layer is then cut in 2 mm2 sections and placed in media for transport to our lab.
- A 60mm2 TCPS plate is scored using a sharp scalpel in several grid patterns. A 2 mm2 section is placed at the intersection of lines on the grid pattern so as to allow the attachment and guidance of the fibroblasts.
- The sections are allowed to dry under a laminar flow hood for 15 minutes before adding 4 ml of DMEM supplemented with 10% Calf Serum and 1% Pen/Strep.
- The media is changed every 2 days. After 10 days individual fibroblasts may be seen. The plate is grown to confluency, at which point the excised dermis is removed and discarded as biohazardous waste.
- The cells are trypsinized and split several more times, after which they may be used for immediate experimental procedures or may be frozen down with 1 % DMSO and stored for later use.
Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4.
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56.
- Mark Ptashne. A genetic switch. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2004. ISBN:0879697164
- Who has experience with this protocol?