Penicillin g

AMCs were isolated as described in our prior reports [14 , 40 (link)]. Briefly, the amnion (~10 g) was peeled from the chorion layer and rinsed 3 times in sterile Hanks’ Balanced Salt Solution (HBSS; Cat# 21–021-CV, Corning) to remove blood debris. The sample was then incubated with 0.05% trypsin/EDTA (Cat# 25–053-CI, Corning) for 1 hour at 37°C (water bath) to disperse the cells and remove the epithelial cell layer. The membrane pieces were then washed 3 times using cold HBSS to inactivate the enzyme. The washed membrane was then transferred into a second digestion buffer containing Minimum Essential Medium Eagle (Cat# 10–010-CV, Corning), 1-mg/mL collagenase type IV, and 25-μg/mL DNase I and incubated in a rotator at 37°C for 1 hour. The digested membrane solution was neutralized using Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 media (Cat# 16–405-CV, Corning), filtered using a 70-μm cell strainer, and centrifuged at 3000 rpm for 10 minutes. The cell pellet was resuspended in complete DMEM/F12 media supplemented with 5% heat-inactivated FBS (Cat# 35–010-CV, Corning), 100-U/mL penicillin G, and 100-mg/mL streptomycin (Cat# 30–001-CI, Corning); plated at 3–5 million cells per T75; and incubated at 37°C with 5% CO ₂ until they were 80%–90% confluent. AMC purity was confirmed with morphological analysis before passaging to P1s.

Mouse melanoma cell lines SW1 and PVMM were obtained as generous gifts from Dr. Ze’ev Ronai of Sanford Burnham Prebys Medical Discovery Institute and Dr. Serge Y. Fuchs of University of Pennsylvania, respectively. Cells were maintained as monolayers in Dulbecco’s Modified Eagle’s Medium (VWR International), supplemented with 10% v/v fetal bovine serum (FBS, Gibco by Life Technologies), 100 IU of Penicillin G and 100 μg/ml Streptomycin (Corning). The cell lines were tested for mycoplasma contamination with MycoAlert Plus Mycoplasma detection kit (Lonza).