To quantify infectious virus, plaque assays or focus formation assays were performed as previously described.90 (link) Briefly, for plaque assays, BHK-21 cells were seeded in 6-well plates and inoculated with 10-fold serial dilutions of serum or tissue homogenate in in vitro diluent. After 1 h incubation at 37°C, cells were overlayed with 1% immunodiffusion agarose (MP Biomedical) and incubated for another 40–44 h. Afterward, neutral red stain was added to visualize plaques. For focus formation assay, Vero cells were seeded in 96-well plates and inoculated with 10-fold serial dilutions of serum or tissue homogenate for 2 h at 37°C. Cells were overlayed 0.5% methylcellulose in MEM-alpha plus 10% FBS and incubated for 18 h at 37°C. Following fixation with 1% paraformaldehyde (PFA), CHIKV-infected cells were detected using CHK-11 monoclonal antibody88 (link) at 500 ng/mL, and foci were visualized with TrueBlue Peroxidase substrate (SeraCare 5510–0030) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology).
Ctl biospot analyzer
Manufactured by Cellular Technology
Sourced in United States
The CTL Biospot analyzer is a laboratory instrument designed for the quantitative analysis of biological samples. It provides accurate and reliable measurements of various analytes in a wide range of applications. The core function of the CTL Biospot analyzer is to perform sensitive and precise quantitative assessments of target molecules or cellular components within a sample.
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6 protocols using ctl biospot analyzer
1
Quantifying Chikungunya Virus Infectivity
2
FRNT Assay for Chikungunya Virus Antibodies
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For FRNT assays, Vero cells were seeded in 96-well plates. Serum samples were heat-inactivated and serially diluted in DMEM/F12 medium with 2% FBS in 96-well plates. Approximately 100 focus-forming units (FFU) of virus stock (CHIKV strain SL15649) was added to each well and the serum plus virus mixture was incubated for 1h at 37°C. At the end of 1h, medium was removed from Vero cells and serum sample plus virus mixture was added for 2 h at 37°C. After 2 h, sample was removed and cells were overlaid with 0.5% methylcellulose in MEM/5% FBS and incubated 18h at 37°C. Cells were fixed with 1% PFA and probed with 500 ng/mL of anti-CHIKV CHK-11 mAb (Pal et al., 2013 (link)) diluted in 1X PBS/0.1% saponin/0.1% bovine serum albumin (BSA) for 2h at room temperature. After washing, cells were incubated with HRP-conjugated goat anti-mouse IgG for 1.5-2 h at room temperature. After washing, CHIKV-positive foci were visualized with TrueBlue substrate and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology, Cleveland, OH). Percent infectivity was calculated compared to a virus only (no serum) control. The FRNT50 value was defined as the reciprocal of the last dilution to exhibit 50% infectivity.
3
ZIKV Neutralization Assay Protocol
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The FRNT50 assay for ZIKV was performed on NHP serum samples. Briefly, fourfold serially diluted serum samples and ZIKV (strain PRVABC59) (≈100 pfu/ml) were incubated for 1 h at 37 °C and added to Vero cells, with overlay medium added after 1 h. After overnight culture, the 96-well plates were stained with an anti-flavivirus monoclonal antibody (MAB10216, Millipore, Burlington, MA USA) and goat anti-mouse IgG (H + L) horseradish peroxidase (HRP)-conjugated secondary antibody (5220–0341, SeraCare Life Sciences, Milford, MA, USA). TrueBlue Peroxidase Substrate (5510–0030, SeraCare Life Sciences, Milford, MA, USA) was added to the plate and spots counted by CTL Biospot Analyzer and Biospot software (Cellular Technology, Ltd, Cleveland, OH, USA). FRNT50 titres were calculated by interpolation between the two points that spanned 50% neutralisation.
4
Quantifying Chikungunya Virus Infectivity
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To quantify infectious virus, plaque assays or focus formation assays were performed as previously described.90 (link) Briefly, for plaque assays, BHK-21 cells were seeded in 6-well plates and inoculated with 10-fold serial dilutions of serum or tissue homogenate in in vitro diluent. After 1 h incubation at 37°C, cells were overlayed with 1% immunodiffusion agarose (MP Biomedical) and incubated for another 40–44 h. Afterward, neutral red stain was added to visualize plaques. For focus formation assay, Vero cells were seeded in 96-well plates and inoculated with 10-fold serial dilutions of serum or tissue homogenate for 2 h at 37°C. Cells were overlayed 0.5% methylcellulose in MEM-alpha plus 10% FBS and incubated for 18 h at 37°C. Following fixation with 1% paraformaldehyde (PFA), CHIKV-infected cells were detected using CHK-11 monoclonal antibody88 (link) at 500 ng/mL, and foci were visualized with TrueBlue Peroxidase substrate (SeraCare 5510–0030) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology).
5
Quantifying Chikungunya Virus Infection
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As previously described [87 (link)], Vero cells were seeded in 96-well plates and inoculated with 10-fold serial dilutions of samples for 2 h at 37°C. Cells were overlayed with 0.5% methylcellulose in MEM-alpha plus 10% FBS and incubated for 18 h at 37°C. Following fixation with 1% paraformaldehyde (PFA), CHIKV-infected cells were detected using CHK-11 monoclonal antibody [88 (link)] at 500 ng/mL, and foci were visualized with TrueBlue Peroxidase substrate (SeraCare 5510–0030) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology).
6
Quantifying Viral Infectivity via Plaque and Focus Assays
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In some experiments, infectious virus was quantified by plaque assay or focus formation assay, as described previously (Hawman et al., 2017 (link)). In brief, for plaque assays, BHK-21 cells were seeded in a 6-well plate and inoculated with 10-fold serial dilutions of serum or tissue homogenate in in vitro diluent. Following a 1 hr adsorption, cells were overlayed with 1% immunodiffusion agarose (MP Biomedical) and incubated at 37° C for 40–44 hr before staining with neutral red stain to enumerate plaques. For focus formation assays, Vero cells were seeded in a 96-well plate and serial dilutions of serum or tissue homogenate were adsorbed to the cells for 2 hr, after which the inoculum was removed and an overlay of 0.5% methylcellulose in MEM-alpha with 10% FBS was added to the cells. Following an 18 hr incubation at 37° C, the cells were fixed with 1% paraformaldehyde and probed with CHK-11 monoclonal antibody at 500 ng/ml in Perm Wash (1x PBS, 0.1% saponin, 0.1% BSA), followed by a secondary goat anti-mouse IgG conjugated to horseradish peroxidase at 1:2000 in Perm Wash. Foci were visualized with TrueBlue substrate (Fisher) and counted with a CTL Biospot analyzer using Biospot software (Cellular Technology).
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