Atcc ccl 81

A clinical isolate of CHIKV (East/Central/South African genotype) was recovered from a patient’s serum and used for virus propagation by a single passage in C6/36 mosquito cells. The viral titre (10⁷ p.f.u./ml) of CHIKV suspension was established by serial dilution on Vero cells using a plaque assay. Vero (African green monkey kidney cells, ATCC CCL-81) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) as a growth medium or 2% FBS as a maintenance medium. All of the drugs used in this study were purchased from Sigma (Sigma Aldrich) and dissolved in DMSO to a final concentration of less than 1% of the total volume.

Human prostate carcinoma cells (PC-3 and LNCaP) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). PC-3 has high metastatic potential, whereas LNCaP has low metastatic potential. PC-3 cells were maintained as monolayers in DMEM supplemented with 10% fetal bovine serum (FBS), 1% streptomycin–penicillin, and 3.7 g/L sodium bicarbonate (NaHCO₃) as a pH buffer agent. LNCaP cells were maintained in RPMI 1640 containing 10% FBS, 0.1% streptomycin-penicillin, 1 mM sodium pyruvate, 10 mM HEPES, and 2 g/L sodium bicarbonate (NaHCO₃). Vero cells, an African green monkey kidney cell line (ATCC® CCL-81™), were purchased from ATCC and used as a representative normal cell line. Vero cells were cultured in EMEM containing 10% FBS and 1% penicillin/streptomycin. All cell types were grown in a 75 cm³ T flask (Corning, NY, USA) under 95% humidified air with a 5% carbon dioxide (CO₂) incubator at 37°C.

For cell culture and maintenance, normal African green monkey kidney fibroblast cells (Vero; ATCC^® CCL-81™) and immortalized human keratinocyte cell line (HaCaT; CLS 300493) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Invitrogen, Carlsbad, CA, USA), supplemented with 10 % fetal bovine serum (v/v), and 1% Antibiotic-antimycotic (v/v) (Gibco, Invitrogen, CA, USA) at 37 °C in the present of 5% CO_2.To test the cytotoxicity effects of normal cell cultures, Vero and HaCaT cells were seeded approximately 1.5 × 104 cells per well into 96 well-plates (Falcon^® a Corning brand, USA) and incubated for 24 h. All cells were then treated with 0 (distilled water only), 500, 1000, and 1500 μg /mL of crude extract. After 96 h of incubation, the mixing solutions were discarded; then, 4 μg of MTT solution was added and incubated for 3 h. Lastly, MTT solution (Invitrogen, USA) was replaced with 50 μL of dimethyl sulfoxide (Thermo Fisher Scientific, CA, USA) prior to the absorbance measurement at 570 nm by using a Infinite^® M Nano microplate reader (Tecan, Männedorf, Switzerland).

Lab amplified virus stocks were prepared as previously mentioned [15 (link),41 (link)]. The virus was quantified using modified plaque assay for 96 well plate format was performed using Vero cells (ATCC^® CCL-81™). Briefly, starting at 1:100 the virus was double diluted till 1:102400. The virus was incubated on the cells for 1–2 h at 37 °C for virus adsorption. Thereafter, the viruses were removed, and wells were overlaid with 150 μL of 1% carboxymethylcellulose (CMC) prepared in sera free DMEM media (i.e., overlay media). The plates were incubated at 37 °C for 48–72 h at 37 °C with 5% CO₂ and 75% humidity. Post incubation the cells were fixed with 10% formaldehyde before washing twice with 1× PBS and staining with 0.25% crystal violet (prepared in 30% methanol). The stained wells were washed with 1× PBS. Virus titers was calculated using the following formula:
Further, viral characterization was performed using Viral nucleic acid (vRNA), isolated from viral stock using High Pure Viral Nucleic Acid Kit (Roche, Grenzach-Wyhlen, Germany) and estimation of viral copy number was done using the QuantiTect reverse transcription kit (Qiagen, Hilden, Germany) as per previously established protocols [15 (link)].