The day before infection, 1×104 Vero E6 cells per well were seeded in an eight well chamber slide (Nunc Labtek chamber slides, Thermoscientific). On the day of infection, SARS-CoV-2, isolate USA-WA/2020 (BEI resources, cat no. NR-52281) was added to cells at an MOI of 0.01. The infection was carried out for 48 hours. Cell layers were washed and incubated with 8 μg/mL A568-conjugated monoclonal antibody for 30 minutes at room temperature (RT), washed and fixed in 10% neutral-buffered formaldehyde for 30 minutes at room temperature (Duke GHRB SOP 38, attachment 21). After two washes with Wash Buffer (1%FBS-PBS; WB), cells were permeabilized with 0.5% Triton for 5 minutes at RT. Blocking buffer was added and the cells were incubated at 4°C for 4 hours. After washing, 10 μg/mL anti-SARS-COV-2 nucleocapsid antibody (40143-MM08, Sino Biological), conjugated with A488 using an Alexa fluor antibody labelling kit (Invitrogen) was added and incubated at 4°C overnight with rocking. Cells were then washed and Fluorshield DAPI reagent (Sigma) added before the cover slip was mounted on the slide. Cells were visualized using a Keyence BZ-X710 fluorescent microscope and images analyzed using BZ-X Analyzer software (v1.3.11).
Fluorshield dapi reagent
Manufactured by Merck Group
Fluorshield DAPI reagent is a laboratory product manufactured by Merck Group. It is a fluorescent stain used for the detection and visualization of DNA in biological samples. The reagent binds to DNA and emits fluorescence under ultraviolet or blue light, allowing researchers to identify and analyze DNA-containing structures within cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor antibody labelling kit, by Thermo Fisher Scientific (4 mentions)
Bz x710 fluorescent microscope, by Keyence (4 mentions)
Nunc lab tek chamber slide, by Thermo Fisher Scientific (2 mentions)
A488 conjugated anti human igg fc antibody, by BioLegend (2 mentions)
Anti sars cov 2 nucleocapsid antibody, by Sino Biological (2 mentions)
Bz x analyzer software, by Keyence (1 mentions)
4 protocols using fluorshield dapi reagent
1
SARS-CoV-2 Infection of Vero E6 Cells
2
SARS-CoV-2 Antibody Detection Assay
3
SARS-CoV-2 Infection and Immunofluorescence Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The day before infection, 1 × 104 Vero E6 cells per well were seeded in an eight well chamber slide (Nunc Labtek chamber slides, Thermoscientific). On the day of infection, SARS‐CoV‐2, isolate USA‐WA/2020 (BEI resources, cat no. NR‐52281) was added to cells at an MOI of 0.01. The infection was carried out for 48 h. Cell layers were washed and incubated with 8 μg/ml A568‐conjugated monoclonal antibody for 30 min at room temperature (RT), washed and fixed in 10% neutral‐buffered formaldehyde for 30 min at room temperature (Duke GHRB SOP 38, attachment 21). After two washes with Wash Buffer (1%FBS‐PBS; WB), cells were permeabilized with 0.5% Triton for 5 min at RT. Blocking buffer was added and the cells were incubated at 4°C for 4 h. After washing, 10 μg/ml anti‐SARS‐COV‐2 nucleocapsid antibody (40143‐MM08, Sino Biological), conjugated with A488 using an Alexa fluor antibody labelling kit (Invitrogen) was added and incubated at 4°C overnight with rocking. Cells were then washed and Fluorshield DAPI reagent (Sigma) added before the cover slip was mounted on the slide. Cells were visualized using a Keyence BZ‐X710 fluorescent microscope and images analyzed using BZ‐X Analyzer software (v1.3.11).
4
SARS-CoV-2 Antibody Detection Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infected cells were prepared as described above. On the day of assay, Cell layers were washed and incubated with plasma from infected individuals diluted at 1:100 for 30 min at room temperature (RT), washed and fixed in 10% neutral‐buffered formaldehyde for 30 min at room temperature (Duke GHRB SOP 38, attachment 21). After two washes with Wash Buffer (1%FBS‐PBS; WB), cells were permeabilized with 0.5% Triton for 5 min at RT. Blocking buffer was added and the cells were incubated at 4°C for 4 h. After washing, 10 μg/ml anti‐SARS‐COV‐2 nucleocapsid antibody (40143‐MM08, Sino Biological), conjugated with A568 using an Alexa fluor antibody labelling kit (Invitrogen), was added and incubated at 4°C overnight with rocking. After washing, A488‐conjugated anti‐Human IgG Fc antibody (Clone: HP6017, Biolegend) was added and incubated for 30 min at 4°C on a rocking platform. Cells were then washed and Fluorshield DAPI reagent (Sigma) added before the cover slip was mounted on the slide. Cells were visualized using a Keyence BZ‐X710 fluorescent microscope and images analyzed using BZ‐X Analyzer software (v1.3.11).
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