Mycoblue mycoplasma detector kit

Cancer cell lines: BxPC-3 (pancreas adenocarcinoma, ATCC^® CRL-1687^TM), and HT-29 (colorectal adenocarcinoma, ATCC^® HTB-38^TM) and normal cell line—WI-38 (human lung fibroblasts, ATCC^® CCL-75TM) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). BxPC-3 cells were grown in RPMI-1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) of both antibiotics (streptomycin and penicillin). For the HT-29 cell line, RPMI-1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) of both antibiotics and 1% MEM nonessential amino acids was used to ensure proper cell growth. WI-38 cells were grown in MEM medium supplemented with 10% (v/v) FBS, L-Glutamine, 25 mM Hepes, and 1% penicillin–streptomycin. MycoBlue^TM Mycoplasma Detector kit (Vazyme Biotech, Nanjing, China) was used at least every month for the control of mycoplasma contamination in the cell cultures.
Cells were grown at 37 °C in a humidified atmosphere of 5% CO₂ in the air. The culture medium was changed every 24–48 h. Subculture was performed using 0.25% trypsin/EDTA after cells reached confluence.

BxPC-3 (pancreas adenocarcinoma, ATCC^® CRL-1687^TM), PC3 (prostate cancer, ATCC^® CRL-1435^TM), and WI-38 (human lung fibroblasts, ATCC^® CCL-75^TM) cell lines were obtained from American Type Culture Collection (ATCC, Rockville, USA). BxPC-3 cells were grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) of both antibiotics (streptomycin and penicillin). PC-3 cells were cultured with DMEM-F12 supplemented with 10% (v/v) FBS and 1% (v/v) of antibiotics (streptomycin and penicillin). WI-38 cells were grown in MEM supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) of both antibiotics (streptomycin and penicillin).
Cells were grown at 37 °C in a humidified atmosphere of 5% CO₂ in the air. The culture medium was changed every 24–48 h. Subculture was performed using 0.25% trypsin/EDTA after cells reached confluence.
MycoBlue^TM Mycoplasma Detector kit (Vazyme biotech Co., Ltd.) was used at least every month for the control of mycoplasma contamination in the cell cultures.
The cytotoxic activity of the pyrazolo[4,3-e][1,2,4]triazines investigated in this study was recently published by our group [19 (link)].

K562 cell line was purchased from the American Type Culture Collection and maintained in RPMI1640 (Thermo Scientific) plus 10% fetal bovine serum (ExCell Bio) and penicillin–streptomycin (Thermo Scientific). Mycoplasma testing of cell culture was performed routinely using a MycoBlue Mycoplasma Detector Kit (Vazyme).

The human PDAC cell line, CFPAC-1, was acquired from the American Type Culture Collection, and cell line authentication was confirmed using Promega GenePrint 10 system. The CFPAC-1 cell line was transduced with oligos and corresponded to the sgRNA sequence (Supplementary Table 1) of sgDCK#10 and control sgNT1 described in our previous article and named as DCK#10 and NT1 cell [10 (link)]. CFPAC-1, DCK#10 (DCK knockout CFPAC-1 cell), NT1 (CFPAC-1 cell with the non-target gRNA insert), HA-DCK (DCK#10 with stable expression of HA-DCK [10 (link)]), HA-DCK-KD (DCK#10 with stable expression of HA-DCK bearing the kinase-dead mutation [10 (link)]) and EV (DCK#10 with stable expression of empty vector [10 (link)]) cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; FUJIFILM Wako Pure Chemical Corporation [FUJIFILM-Wako]), HPAF-II cells were maintained in Eagle’s minimum essential medium (FUJIFILM-Wako). All cell culture media were supplemented with 10% heat-inactivated FBS (Gibco #10270–106) and 1% penicillin–streptomycin (10,000 U/mL; Thermo Fisher Scientific, #15140–122). Cells were cultured in a humidified atmosphere containing 5% CO₂ at 37 °C and maintained in culture for less than 20 passages. Mycoplasma testing was conducted using MycoBlue mycoplasma detector kit (Vazyme).

OC OVCAR3 (ATCC HTB-161), SKOV3 (ATCC HTB-77), HO-8910, and PEO1 cells were grown in RPMI 1640 media supplemented with 10% v/v heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 1% w/v glutamine and 1% w/v antibiotics. All the cell lines involved were cultured at 37 °C in a humidified incubator containing 5% CO₂. Cells were routinely tested for mycoplasma using MycoBlue Mycoplasma Detector kit (Vazyme). Shanghai Biowing Applied Biotechnology (Shanghai, China) characterized short tandem repeat (STR) profiling to confirm the cell lines authenticity.

SW13, HEK293T, Vero E6 (all from ATCC), Huh7, HUVEC (provided by National Virus Resource Center, Wuhan, China) and DLD1 (provided by Dr. Youjun Li, Wuhan University) cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (Hyclone). The primary mouse hepatocytes and MLF cells were isolated and cultured as previously described.^{55 (link),56 (link)} Primary human PBMCs were isolated from whole blood of healthy donors with SepMate™-15 (STEMCELL Technologies) according to the manufacturer’s instructions and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The isolation of human PBMCs and its application for virus infection were approved by Institutional Review Board, Wuhan Institute of Virology, Chinese Academy of Sciences (Approval Number: WIVH31202301). FreeStyle 293F cells (provided by Dr. Bing Yan, Wuhan Institute of Virology, Chinese Academy of Sciences) were cultured at 37 °C in SMM 293-TII Expression Medium (SinoBiological) on an orbital shaker platform rotating at 120 rpm. All cell lines were tested and found to be free of mycoplasma contamination using the MycoBlue Mycoplasma Detector kit (#D101, Vazyme).