FluoroSpot Assay for ASCs

Renata Varnaitė; Sara Gredmark-Russ

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FluoroSpot Assay for ASCs

RV Renata Varnaitė

SG Sara Gredmark-Russ

Last updated date: Jun 28, 2021 Views: 792 Forks: 0

An abbreviated version of this protocol was published in J Immunol in Sep, 2020

Expansion of SARS-CoV-2–Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients

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Human IgG/IgM/IgA FluoroSpot – SARS-CoV-2 nucleocapsid protein specific and total ASC numbers in peripheral blood of COVID-19 patients

Reagents

Human IgG/IgM/IgA Fluorospot Kit	Mabtech	X-06G05R17M-10
Human IgG set (550) Human IgA set (490) Human IgM set (640) Fluorospot plates R848 rIL-2 Fluorescence enhancer SARS-CoV-2 nucleocapsid protein (made in-house) U bottom 96 well plate 99.99% Ethanol MilliQ H₂O R10 media dPBS	10 IPFL plates	+4° +4° +4° RT -20° -20° +4° -80°
	Diluted to 35% in sterile water

	RPMI 1640 with 10% FBS, 1% Pen/Strep, 1%L-Glutamine

Protocol

Coating of plate with anti-IgG/IgA/IgM or SARS-CoV-2 antigen: Can prepare up to 7 days prior

In the same tube dilute the capture mAbs to 15µg/mL for total IgA/G/M wells
Pre-wet the plate membrane by treatment with 35% ethanol, 15µl per well for max 1 minute
Wash the plate 5 times with sterile H₂O, 200µl/well
Add 100µl/well of capture antibody solution, antigen solution or PBS according to layout below and incubate at least overnight at 4-8°C
Prepare R10 media

Incubation of cells on plate:

Remove excess antibody and wash the plate 5 times with sterile PBS, 200µl/well
Throw PBS from plate and add 200µl/well of sterile R10 and incubate at least 30 minutes to 1hour at RT to block/condition the membrane
Prepare cell dilution plate after cells are counted
1. Move at least 1 million cells to FACS tube with lid and wash in R10 (1500 rpm 5 min)
2. Resuspend to 1 million cells/100µl as Stock Cells and dilute as follows in DILUTION PLATE: (number in well denotes number of cells added per well in thousands)

DILUTION PLATE (numbers in wells denote number of cells added in thousands)

µl of R10	72	96	108	108	117.6	117.6	72	96	108	108	117.6	117.6
µl Stock Cells	48	24	12	12	2.4	2.4	48	24	12	12	2.4	2.4
	1	2	3	4	5	6	7	8	9	10	11	12
A	480	240	120	120	24	24	480	240	120	120	24	24
B
C	480	240	120	120	24	24	480	240	120	120	24	24
D
E	480	240	120	120	24	24	480	240	120	120	24	24
F
G	480	240	120	120	24	24	480	240	120	120	24	24
H

4. Remove the medium from FLUOROSPOT PLATE after blocking and add 50µl of R10 to each well according to plate layout on next page

Move 50µl of cells from DILUTION plate (above) to FLUOROSPOT plate (see plate layout below)

5. Place plate in 37°C humidified incubator with 5% CO2 and incubate 16-24 hours. Do no move the plate during this time and wrap plate in aluminum foil to avoid evaporation

FLUOROSPOT PLATE (numbers in wells denote number of cells added in thousands)

	SARS-CoV-2 Ag 10µg/ml			IgG/IgM		PBS	SARS-CoV-2 Ag 10µg/ml			IgG/IgM		PBS
	1	2	3	4	5	6	7	8	9	10	11	12
A	200	100	50	50	10	10	200	100	50	50	10	10
B	200	100	50	50	10	10	200	100	50	50	10	10
C	200	100	50	50	10	10	200	100	50	50	10	10
D	200	100	50	50	10	10	200	100	50	50	10	10
E	200	100	50	50	10	10	200	100	50	50	10	10
F	200	100	50	50	10	10	200	100	50	50	10	10
G	200	100	50	50	10	10	200	100	50	50	10	10
H	200	100	50	50	10	10	200	100	50	50	10	10

Detection of spots:

Wash the plate 5 times with PBS, 200µl/well
Prepare the detection reagents below:

a. Add 100µl/well for 2 hours at RT in dark

3. Wash as in step 1

4. Empty the plate and add 50µl/well of Fluorescence enhancer-II and incubate for 15 minutes at RT

5. Empty plate and firmly removed enhancer by firmly tapping the plate against paper towels

6. Remove underdrain and leave in dark to dry. Inspect and count spots in Mabtech IRIS FluoroSpot reader.

Store plate in dark at RT

Related files

ASC FluoroSpot SARS-CoV2.docx download

How to cite：

Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:

Varnaitė, R and Gredmark-Russ, S(2021). FluoroSpot Assay for ASCs. Bio-protocol Preprint. bio-protocol.org/prep1222.
Varnaitė, R., García, M., Glans, H., Maleki, K. T., Sandberg, J. T., Tynell, J., Christ, W., Lagerqvist, N., Asgeirsson, H., Ljunggren, H., Ahlén, G., Frelin, L., Sällberg, M., Blom, K., Klingström, J. and Gredmark-Russ, S.(2020). Expansion of SARS-CoV-2–Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients. J Immunol 205(9). DOI: 10.4049/jimmunol.2000717