Mycosensor pcr assay kit

HCT116, THP-1 and RAW 264.7 Cells were procured from ATCC (Manassas, VA, USA). HCT116 and THP-1 cells were cultured in DMEM and RPMI, respectively, with 10% fetal bovine serum. RAW 264.7 cells were grown in DMEM without phenol red, containing media with 10% fetal bovine serum and antibiotic-antimycotic solution. Cells were routinely checked for mycoplasma contamination using the MycoSensor PCR Assay Kit, from Agilent (Santa Clara, CA, USA).

ZIKV stocks were grown in Vero cells. Cell supernatant was collected when cells showed >80% cytopathic effect. Supernatants were cleared of cell debris (5000× g, 5 min, 4 °C), aliquoted into cryovials, and frozen at −80 °C for one week before titers were determined. The amount of infectious virus was quantified by either TCID₅₀ or plaque assays. African isolate MR766 was derived from the original ZIKV isolation and underwent extensive passaging in both mice and tissue culture cells. African isolate IbH 30656 (VR-1829™, ATCC) was also extensively passaged in cell culture before it was obtained. The IbH stock was obtained from ATCC. Asian isolate MEX1-44 was isolated from a mosquito in Chiapas, Mexico, and the virus was obtained after being passaged four times. The lab passaged the virus three additional times in Vero cells before completing these studies. Asian isolate SPH was isolated from a male ZIKV patient in Brazil. The virus was passaged two times in Vero cells before the stock was received, and the virus was passaged three times in Vero cells before performing the experiments. Prior to experimentation, all ZIKV isolates tested negative for Mycoplasma contamination (MycoSensor PCR Assay Kit, Agilent, West Cedar Creek, TX, USA).

MDCK cells were generously provided by Prof. Jianzhu Chen (MIT), and were originally purchased from American Type Culture Collection (Manassas, VA). The identity of these cells was authenticated by STR profiling. MDCK cells were cultured at 37°C in a 5% CO₂ atmosphere in DMEM (CellGro) supplemented with 10% fetal bovine serum (CellGro) and 1% penicillin/streptomycin/glutamine (CellGro). Cells were transduced with lentiviruses encoding either the DHFR.HSF1(Δ186–202) or DHFR.YFP gene. Heterostable cells expressing the construct of interest were then selected using 4 μg/mL puromycin. Single colonies were generated by diluting cells to ~40 cells per 96-well plate, expanding the resulting colonies, and functionally testing by qPCR or fluorescence microscopy in the presence or absence of trimethoprim (TMP; 10 μM). All cell lines were periodically tested for mycoplasma using the MycoSensor PCR Assay Kit from Agilent (302109).