I am doing plaque assay for CHIK-V virus. After incubation plate was wash fix with 3.7% formaldehyde sol. After fixing cell was detected. Before fixing monolayer was fine.
Hello Parveen Kumar
Use 5-7% formalin for fixation for a minimum of 30 mins to 60 mins. Also, the seeding density is important. It could be that the cells are overconfluent at the time of fixation and so likely to detach very easily.
Best Wishes.
Dear Parveen!
Please You look at the articles:
Article Development and optimization of a direct plaque assay for hu...
African green monkey kidney epithelial cells (Vero ATCC no. CCL-81 and Vero E6 ATCC no. CRL-1586)
2.5. Direct plaque assays for rhMPV-F and aMPV
Vero or Vero-E6 cells were seeded in 6-well plates (Corning) at the density of 2×106 cells per well. After incubation for 18 h, the medium was removed and cell monolayers were infected with 400 µl of a 10-fold dilution series of each virus. After incubation at 37°C for 1 h with agitation every 10 min, the cells in each well were overlaid with 2.5 ml of Eagle minimum essential medium (MEM) containing 1% agarose, 1% FBS, 0.075% sodium bicarbonate (NaHCO3), 20mM HEPES (pH 7.7), 2 mM L-Glutamine, 12.5 mg/mL of penicillin, 4 mg/mL of streptomycin, and 4 mg/mL of kanamycin. The plates were incubated at 4 °C for 30 min to solidify the overlay media. Cells were then grown at 37 °C and 5% CO2 to allow for plaque formation. Where indicated, the overlay medium was supplemented with actinomycin-D (Sigma) (0.1 to 0.6 µg/ml) or TPCK-trypsin (0.1 to 0.6 µg/ml). After incubation for 4–10 days, the cells were fixed in 10% (v/v) formaldehyde for 2 h, and the plaques were visualized by staining with 0.05% (wt/vol) crystal violet.
https://www.nature.com/articles/s41598-017-15269-w
Cell culture
All mammalian cell lines were maintained at 37 °C with 5% CO2. Huh7, SVG-A, Dermal Fibroblasts, HepG2, RD, BHK-21, A549, HeLa and Vero E6 cells were maintained in DMEM supplemented with 10% FCS, 0.5 mM non-essential amino acids and penicillin-streptomycin (100 units/mL).
Plaque assays
Cells were washed with PBS and 180 µL of virus dilution added to wells. Plates were rocked for 5 min prior to incubation at 37 °C for 1 h. Virus solution was removed, cells washed in PBS and layered with 2 mL methyl cellulose (MC, 0.8%) in complete media. Cells were then further incubated for 48 h, where MC was removed, cells fixed in 10% formaldehyde for 30 min and stained crystal violet (0.5%) for 30 min. Cells were washed with water until plaques became visible.
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