Mycoalert assay kit

Human osteosarcoma (143B and U-2 OS), human cervical adenocarcinoma (HeLa and HeLa S3), human epithelial lung carcinoma (A549), murine colorectal cancer cell (CT-26.WT), and murine breast carcinoma (4T1) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human lung carcinoma (NCI-H460), human normal lung fibroblast (MRC-5), human kidney carcinoma (A-498 and Caki-1), human colorectal adenocarcinoma (HCT 116), human metastatic breast carcinoma (MDA-MB-231 and MCF7), and murine renal adenocarcinoma (Renca) were obtained from the Korean Cell Line Bank (KCLB, Chongno-gu, Seoul, Korea). All cell lines were maintained with ATCC and KCLB recommended media, respectively, and supplemented with 10% fetal bovine serum (FBS). The NIH TC-adapted WR strain of VV (VR-1354, ATCC; Manassas, VA, USA) was purchased from the ATCC, amplified in HeLa or HeLa S3 cells, and quantified using a vaccinia virus titration protocol. WR-GFP is a tk deleted and green fluorescence protein armed tk-deleted WR VV obtained from Professor Kim Jae-Ho, Pusan National University, South Korea. All cell lines and viruses were cultured under appropriate conditions and were routinely tested for sterility and mycoplasma contamination using MycoAlert assay kit (Lonza, Rockland, ME, USA).

HMEC-1, carrying the type O blood group and thus not reactive with anti-blood group antibodies in human sera, were supplied by ATCC and were cultured in the MCDB 131 medium (Gibco) supplemented with 10% heat-inactivated FCS (Sigma), 2 mM l-glutamine (Gibco), 100 IU ml⁻¹ penicillin (Gibco), 100 μg ml⁻¹ streptomycin (Gibco), 10 ng ml⁻¹ epidermal growth factor (Gibco), and 1 µg ml⁻¹ hydrocortisone (Sigma-Aldrich). HUVECs were established from primary cultures as previously described⁷⁵ and cultured in Medium 199 (Gibco) supplemented with 20% FCS, 2 mM l-glutamine, 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 130 μg ml⁻¹ heparin (Sigma-Aldrich), and 1.2 mg ml⁻¹ endothelial cell growth supplement (Sigma-Aldrich). CHO-K1 and xylotransferase-1-deficient CHO-K1 cells (pgsA-745 cells), which are HS and GAG deficient, were supplied by ATCC and grown in RPMI-1640 medium supplemented with 5% FCS and antibiotics. All cell lines were incubated in 5% CO₂ and ambient O₂ at 37 °C and were repeatedly tested for mycoplasma using a MycoAlert Assay Kit (Lonza).

All parasites and host cells were routinely tested for Mycoplasma contamination by using the MycoAlert assay kit (Lonza, Basal, Switzerland).