Mycoplasma detection kit

NALM6 and REH parental cell lines were purchased from the American Type Culture Collection (ATCC) and cultured according to the manufacturer’s instructions in RPMI 1640 media supplemented with 10% FBS and 1% penicillin/streptomycin. WT Raji and CD19-KO Raji (CRISPR/Cas9) cell lines were generated at the Icahn School of Medicine at Mount Sinai. All cell lines were tested for mycoplasma using a mycoplasma detection kit (InvivoGen). NALM6, and REH parental cells were exposed to HD37-dgRTA anti-CD19 immunotoxin (11 (link)) at IC₁₀ (5 × 10^–13 M) for 7 days. The dose was escalated as shown in Figure 1A for a total of 30 days of treatment. Once resistant cells were established and in culture, they were treated every 2–3 days with immunotoxin at the IC₇₅ dose to maintain resistance.

The ER + MCF7 and T47D human breast cells, which were all obtained from ATCC, authenticated using STR profiling with CellCheck 9 Plus by IDEXX Bioresearch, and tested for mycoplasma using Mycoplasma Detection Kit, InvivoGen, Inc in April 2016, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and Antimycotic solution (Sigma). Cells were maintained in a humidified 37 °C environment with 5% CO₂. pMEFs were isolated from E14.5 isogenic SIRT3^+/+ mice (through a protocol that was approved by the Institutional Animal Care and Use Committee (IACUC) and complied with related animal research ethical regulations) and maintained in a 37 °C incubator with 5% CO₂ and 6% oxygen, except when otherwise noted. MCF7 and T47D cells were grown for 3 months in 1 μM hydroxy-Tam to create MCF7-HTR and T47D-HTR permanent cell lines, and several different subclones were frozen. MCF7-HTR and T47D-HTR were not used for >5 passages, and new cell lines were used. All experiments were done using exponentially growing cell cultures at 50% confluence.

Human pancreatic cancer cell lines Panc0203, Panc1, Panc0403, Capan1, MiaPaCa2, and BxPC3 were obtained from the ATCC. Panc0203, Panc0403, Capan1, and BxPC3 cells were cultured in RPMI1640 whereas Panc1 and MiaPaCa2 cells were cultured in DMEM (Gibco, Thermo Fisher Scientific). All culture media were supplemented with 10% heat-inactivated FBS (R&D Systems), and 1% penicillin-streptomycin (Sigma). Cells were grown at 37°C with 5% CO₂. Cell lines were used between passage 3 and 5 after they were taken out of the liquid nitrogen. Cell lines were authenticated by the University of Arizona Genetics Core. All cell lines were Mycoplasma free, monitored regularly with HEK-blue2 cells and Mycoplasma Detection Kit from InvivoGen (catalog no. rep-pt1).