Heat inactivated fetal bovine serum

Baby hamster kidney (BHK-21) cells (ATCC no. CCL-10) were maintained in Glasgow minimum essential medium (GMEM) (Life Technologies, USA) supplemented with 5% heat-inactivated fetal bovine serum (Bovogen Biologicals, Australia), 10% Tryptose phosphate broth (Sigma-Aldrich, USA), 20 mM HEPES, 5mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). Infected cells were maintained in GMEM containing 2% FBS.
The virus strain used was MY/08/065 (GenBank accession number FN295485) at passage 3, a previously characterized ECSA virus isolated from a patient in Malaysia in 2008 [30 (link)]. The virus was propagated in BHK-21 cells and titrated by standard plaque assay. To study the neutralizing epitopes of anti-CHIKV IgM and IgG, CHIKV and previously constructed chimeric viruses carrying zsGreen reporter were rescued from infectious clones, as previously described [27 (link)]. The CHIKV infectious clone was derived from the ECSA genotype, based on LR2006-OPY1 and termed ICRES1 [31 (link)], and the chimeric viruses had the ecto-domain regions of envelope glycoproteins in the ICRES1 backbone replaced with those of Semliki Forest virus (SFV), a related alphavirus [27 (link)]. The chimera with E1 swapped from SFV was non-viable.

NSC-34 (86 (link)) cells were cultured in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) (Invitrogen), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Bovogen). In order to passage and plate NSC-34 cells, they were washed once with prewarmed DMEM-F12 and treated with 0.25% trypsin and 0.02% EDTA dissociation reagent (Invitrogen) to lift off the adherent cells. The cells were pelleted via centrifugation (300g for 5 min) and resuspended in prewarmed culture media. Following washing, plates were seeded at a confluency of 40% and cultured at 37 °C in a humidified incubator with 5% atmospheric CO₂ for 24 h prior to transfection (∼70–80% confluent). Cells were transfected with plasmid DNA (0.5 μg per well of a 24-well plate, 2.5 μg per well of a 6-well plate) 24 h postplating using TransIT-X2 reagent (Mirus Bioscience) according to the manufacturer's instructions.

Human SH-SY5Y neuroblastoma cells were grown and sustained in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, Logan UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (Bovogen Biologicals, Essendon, VIC, Australia), 2% HEPES (Gibco, Carlsbad, CA, USA) and 1% L-glutamine (Invitrogen, Carlsbad, CA, USA). Cultures were maintained at 37 °C in a 95% oxygen, 5% carbon dioxide, 90% humidity atmosphere and passaged regularly via washing with phosphate buffered saline (PBS; Gibco) and trypsinization (0.1% trypsin-EDTA in PBS; Gibco). All cells used in this study were passage 7 at the time of harvest.