E myco plus mycoplasma pcr detection kit

Cell lines B7E3, B4B8, and B7E11 were obtained from Dr. Carter Van Waes [26 (link)]. All cell lines were grown and maintained in high-glutamine DMEM with the following supplements: 10% FBS, 1% nonessential amino acids, and antibiotics. All cell lines were tested by the eMycoPlus Mycoplasma PCR detection kit (BulldogBio) to ensure they were bereft of any mycoplasma infection.

The UM-SCC-104 (referred to as SCC104) cell line was obtained from Sigma-Aldrich, and the UPCI : SCC152 cell line (referred to as SCC152) was obtained from ATCC. Both SCC104 and SCC152 cell lines have been reported to be HPV-16 positive (24 (link), 25 (link)). SCC25 and SCC47 cell lines were purchased from ATCC and Millipore Sigma, respectively. Cell lines UM-SCC-11B, UM-SCC-74A, UM-SCC-29, UM-SCC-23, and UM-SCC-103 were obtained from Dr. Thomas Carey (University of Michigan). HSC-3 and CAL-27 cell lines were generously provided by Dr. Manish Bais (Boston University). All cell lines were grown and maintained in high-glutamine DMEM or DMED/F12 as recommended, with the following supplements: 10% FBS, 1% nonessential amino acids, and antibiotics. Other cell lines used in this study have been described before in Gluck et al. 2019 (26 (link)). The identities of the cell lines utilized in this study were confirmed via short tandem repeat profiling through services offered by Genetica. All cell lines were tested by the eMycoPlus Mycoplasma PCR Detection Kit (BulldogBio) to ensure that they were bereft of any mycoplasma infection.

PC-3 and DU-145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Both the cell lines were at passage 25 when the experiments were performed. These cells differ in their aggressive behavior significantly, and we have documented this in an earlier publication [34 (link)]. DU-145 was originally isolated from brain metastases and PC-3 was isolated from lumbar vertebrae. Both these cell lines are androgen-independent, and they were grown at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ in RPMI-1640 medium supplemented with non-essential amino acids, l-glutamine, a twofold vitamin solution (Life Technologies, Grand Island, NY, USA), sodium pyruvate, Earle’s balanced salt solution, 10% fetal bovine serum, and penicillin and streptomycin (Flow Labs, Rockville, MD, USA). The cells were harvested by trypsinization. The cell lines used in this study were examined by the eMycoPlus Mycoplasma PCR Detection Kit (BulldogBio, Portsmouth, NH, USA) to confirm that they did not have any mycoplasma infection.

The following cell lines were purchased from American Type Culture Collection (ATCC): DLD-1 (male, adult, age not reported, Dukes’ type C colon cancer), DLD-1 cells were cultured in RPMI 1640 medium (ThermoFisher) supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin. The DLD-1 cells harboring doxycycline-inducible p14^ARF-miRFP670, p14^ARFΔH1-3-miRFP670, miRFP670, were maintained in RPMI 1640 medium supplemented with 10% Tet system approved fetal bovine serum (ThermoFisher), and 250 μg/ml G418. All cell lines were incubated at 37°C in a humidified incubator with 5% CO₂. Gene edited cell lines were authenticated by short tandem repeat (STR) profiling. Cells were tested negative for mycoplasma by the e-Myco PLUS Mycoplasma PCR Detection Kit (Bulldog Bio).