A monolayer of cells in 6-well plates were infected with WI/18 virus at the dilution that gives 10-20 plaques per well for each cell line. Cells were washed with PBS at 1 h post-infection. Cells were covered with an overlay consisting of MEM, 1.2% (w/w) colloidal microcrystalline cellulose, 2 mg mL−1 L-Glutamine, 1 μg mL-1 TPCK-trypsin and incubated for 3 days at 37 °C. After being fixed by 4% PFA and permeabilized with 0.1% Triton-X100, infected cells were stained with anti-NP antibody (1 μg/mL, H16-L10-4R5 (HB-65), BioXcell) and subsequently stained with a secondary HRP-conjugated goat anti-mouse IgG antibody (1:2000, Southern Biotech). Plaques were visualized with KPL TrueBlue (Seracare). Images were acquired with Keyence BZX700 Widefield fluorescence microscope and analyzed using image analysis software ilastik61 (link) and Fiji62 (link).
Bzx700 widefield fluorescence microscope
Manufactured by Keyence
The BZX700 Widefield fluorescence microscope is a laboratory equipment designed for fluorescence imaging. It provides high-resolution, wide-field observation of fluorescently-labeled samples.
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Lab products found in correlation
Hrp conjugated goat anti mouse igg antibody, by Southern Biotech (1 mentions)
Kpl trueblue, by LGC (1 mentions)
Sc 7294, by Santa Cruz Biotechnology (1 mentions)
Ab62613, by Abcam (1 mentions)
Sml1558, by Merck Group (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
A21121, by Thermo Fisher Scientific (1 mentions)
Sc 8405, by Santa Cruz Biotechnology (1 mentions)
2 protocols using bzx700 widefield fluorescence microscope
1
Plaque Assay for Influenza Virus
2
PIEZO1 Agonist Regulation of NFAT Signaling
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After the cells attached to 96-well plate, DMSO or PIEZO1 agonist (Yoda1, 25 μM, SML1558, Sigma-Aldrich) treatment was performed. After 4 h, cells were fixed with 4% Paraformaldehyde/ phosphate-buffered saline (PFA/PBS) for 15 min. The cells were blocked with 5% bovine serum albumin (BSA) and 0.8% Triton X-100 in PBS for 2 h at 16 °C. Cells were incubated with primary antibodies in 1% BSA, 0.8% Triton X-100, 10% DMSO in PBS at 4 °C overnight. After the cells were washed with PBS three times for 5 min each, they were incubated with secondary antibodies for 1 h at 16 °C. After washing with PBS three times for 15 min, we evaluated the cells using a Keyence BZX700 widefield fluorescence microscope. The antibodies used in this experiment were as follows: Anti-NFATC1 (1:100, sc-7294, Santa Cruz), Anti-NFATC2 (1:100, 8032S, Cell Signaling), Anti-NFATC3 (1:100, sc-8405, Santa Cruz), Anti-NFTAC4 (1:100, ab62613, Abcam), Alexa Fluor 488 conjugate (1:500, A11034 and A21121, Invitrogen, B13423), and Hoechst (1:5000, 33342, Invitrogen). ImageJ software was used to measure cell signal intensity (these were measured three times using different views).
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