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).
Immunodiffusion agarose
Manufactured by MP Biomedicals
Sourced in United States
Immunodiffusion agarose is a specialized gel medium used for the detection and identification of proteins through the process of immunodiffusion. It serves as a substrate for antigen-antibody reactions, enabling the visualization and analysis of precipitation patterns formed during this immunological interaction.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Thermo Fisher Scientific (3 mentions)
Formaldehyde, by Thermo Fisher Scientific (3 mentions)
Methanol, by Thermo Fisher Scientific (3 mentions)
Trueblue peroxidase substrate, by LGC (2 mentions)
Biospot software, by Cellular Technology (2 mentions)
Ctl biospot analyzer, by Cellular Technology (2 mentions)
Six well plate, by Corning (2 mentions)
Vero e6, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
6 protocols using immunodiffusion agarose
1
Quantifying Chikungunya Virus Infectivity
2
Viral Plaque Assay Protocol
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Ten-fold serial dilutions of virus stock were prepared in virus dilution medium and were added, in duplicate, to confluent Vero-E6 monolayers in six-well plates (Corning; 200 μl/well). After 1 hour of incubation at 37 °C, 5% CO2, 2 ml/well of virus growth medium containing 0.1% (w/v) immunodiffusion agarose (MP Biomedicals) was added and incubation was continued for 96 hours. Plates were fixed with 2 ml/well formaldehyde (37% (w/v) formaldehyde stabilized with 10-15% (v/v) methanol; Fisher Scientific) for 15 minutes at room temperature. Agarose and fixative were discarded and 1 ml/well 1% (w/v) crystal violet in 10% (v/v) methanol (both Fisher Scientific) was added. Plates were stored at room temperature for 20 minutes and then rinsed thoroughly with water. Plaques were then enumerated.
3
Viral Plaque Assay Protocol
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Tenfold serial dilutions of virus stock were prepared in virus dilution medium and were added, in duplicate, to confluent Vero E6 monolayers in six-well plates (Corning; 200 µl/well). After 1 h of incubation at 37 °C, 5 % CO2, 2 ml/well of virus growth medium containing 0.1 % (w/v) immunodiffusion agarose (MP Biomedicals) was added and incubation was continued for 96 h. Plates were fixed with 2 ml/well formaldehyde [37 % (w/v) formaldehyde stabilized with 10–15 % (v/v) methanol; Fisher Scientific] for 15 min at room temperature. Agarose and fixative were discarded and 1 ml/well 1 % (w/v) crystal violet in 10 % (v/v) methanol (both Fisher Scientific) was added. Plates were incubated at room temperature for 20 min and then rinsed thoroughly with water. Plaques were then enumerated.
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
Plaque Assay for SARS-CoV-2 Virus
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Samples were prepared in Opti-MEM (Gibco) and were added, in duplicate, to confluent Vero E6 monolayers in six-well plates (200 μl per well; Thermo Fisher Scientific). After 1 hour of incubation at 37°C, 5% CO2, virus growth medium (2 ml per well) containing 0.1% (w/v) immunodiffusion agarose (MP Biomedicals) was added and incubation was continued for 72 hours. Plates were fixed with formaldehyde (2 ml per well) [37% (w/v) formaldehyde stabilized with 10 to 15% (v/v) methanol; Thermo Fisher Scientific] for 15 min at room temperature. Agarose and fixative were discarded, and 1% (w/v) crystal violet (1 ml per well) in 10% (v/v) methanol (both Thermo Fisher Scientific) was added. Plates were incubated at room temperature for 20 min and then rinsed thoroughly with water. Plaques were then enumerated.
6
BHK Cell Plaque Assay Protocol
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Plaque sizes were measured during a standard BHK cell plaque assay using 0.5% immunodiffusion agarose (MP Biomedicals Irvine, CA, USA) for overlay. Plaque assay plates were stained with neutral (as described in [21 (link)]) followed by inversion over a light box and measurement of plaque diameters with a metric ruler. Sixty plaques across three wells were counted for each virus and each experiment was performed twice with similar results.
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