FluoroSpot Plus:
Human IFN-γ/IL-4
FSP-0116-2
FSP-0116-10

www.mabtech.com
The products are for research use only.
MABTECH shall not be liable for the use or handling of the product or for consequential, special, indirect or incidental
damages therefrom.

INTRODUCTION
FluoroSpot – General description
This dual FluoroSpot kit enables convenient enumeration of cells secreting either one orboth analytes.
FluoroSpot combines the sensitivity of ELISpot with the capacity to analyze secretion ofseveral analytes simultaneously. This highly sensitive cellular assay is robust, easy to perform, and suitable both for testing single samples and for large-scale screening.

Anti-tag mAb-490
SA-550TAG
Detection mAb-tag
Analyte 1
Capture mAb

BDetection mAb-biotin
Analyte 2
Capture mAb

Our FluoroSpot Plus kits include pre-coated plates ready to use. Monoclonal antibodies(mAbs) are coated onto a PVDF membrane in a 96-well plate. If several analytes areanalyzed, mAbs with different specificities are coated. Detection of cells secreting single
or multiple analytes is made possible using biotinylated and/or tag-labeled detection
mAbs. The detection step is visualized and amplified by specific fluorophore-conjugatedreagents and the resulting spots are analyzed in an automated reader.

2CONTENTS AND STORAGE
FluoroSpot Plus: Human IFN-γ/IL-4
The kit is shipped at ambient temperature. All reagents should be stored at 4-8°C upon
arrival. Plates should be stored at room temperature. The expiry date indicates how longunopened products, stored according to instructions, are recommended to remain in use.
ComponentDetection mAbs

2-plate kit

10-plate kit

120 μl600 μl80 μl400 μlAnti-BAM mAb, 490

120 μl600 μl

SA-550120 μl600 μlAnti-IFN-γ mAb (7-B6-1), BAM
Anti-IL-4 mAb (IL4-II), biotin

Fluorophore conjugates

Concentration

1 mg/ml

Co-stimulus

Anti-CD28 mAb (CD28-A)

0.1 mg/ml

100 μl500 μlPositive control

Anti-CD3 mAb (CD3-2)

0.1 mg/ml

100 μl100 μlBuffer/Solution

FluoroSpot enhancer

25 ml

60 ml

Pre-coated plates

Plates pre-coated with:

210mAbs 1-D1K and IL4-I

Anti-CD28 and anti-CD3 mAbs are supplied in sterile filtered (0.2 μm) PBS. The biotinylated
antibody contains 0.02% sodium azide. The BAM-conjugated antibody, fluorophore-conjugated reagents, and the FluoroSpot enhancer contain 0.002 % Kathon CG. Vials have been
overfilled to ensure recovery of stated volume.

3

ASSAY PROTOCOL
Please consult the table below for recommendations on dilutions and cell incubation time.
The reagents should not be diluted in advance. For additional information, read the Tips
For A Successful Assay section.
ComponentDilution

Step

Anti-IFN-γ mAb (7-B6-1), BAM

1:200C2

Anti-IL-4 mAb (IL4-II), biotin

dilute to 3

C2

Anti-BAM mAb, 490

1:200

C4SA-550

1:200C4Co-stimulus

Anti-CD28 mAb (CD28-A)

1:1000

B1

Positive control

Anti-CD3 mAb (CD3-2)

1:1000

B1

Detection mAbs

Fluorophore conjugates

Cell incubation time
18-48 hours

μg/ml

B2

A. Plate preparation (sterile conditions)
1. Remove the plate from the sealed package and wash the plate three times withsterile PBS (200 µl/well).
2. Block/condition the plate by adding cell incubation medium containing 10% fetal
calf serum (200 µl/well). Incubate for at least 30 minutes at room temperature.

B. Cell incubation (sterile conditions)
1. Remove the medium and add the stimuli followed by the cell suspension. Cells andstimuli can also be mixed before addition to the plate.
2. Incubate the cells in the plate at 37 °C in a humidified incubator with 5% CO2.
Approximate incubation time is indicated above and can be further optimizedby the user. Do not move the plate during incubation and avoid evaporation, forexample by wrapping the plate in aluminum foil.

4C. Detection
1. Remove the cells by emptying the plate and then wash the plate five times withPBS (200µl/well).
2. Dilute the detection antibodies in PBS containing 0.1% BSA (PBS-0.1% BSA)
according to the table above. If more than one analyte is analyzed, dilute thedetection antibodies in the same tube. Add 100 µl/well and incubate for 2 hours atroom temperature.
3. Wash the plate five times with PBS (200 µl/well).
4. Dilute the fluorophore conjugates in PBS-0.1% BSA to the concentrations as shownin the table above. If more than one analyte is analyzed, dilute the conjugatesin the same tube. Add 100 µl/well and incubate for 1 hour at room temperature.
Protect the plate from light throughout the assay.
5. Wash the plate five times with PBS (200 µl/well).
6. Empty the plate, add FluoroSpot enhancer (50 µl/well), and leave for 5-15 minutesat room temperature.
7. Empty by flicking the plate to remove the FluoroSpot enhancer. Do not wash. It is
important that the plate is not tapped against paper towels since dust particles
may interfere with the assay.
8. Remove the underdrain (the soft plastic drain under the plate), for example using
pliers. Dry the plate protected from light. The plate should be completely dry
before analysis. Store plate in the dark at room temperature.
9. Spot analysis is performed with an automated FluoroSpot reader equipped withfilters for the fluorophores used. Filters should have high specificity to avoid bleedthrough artifacts.

AnalyteRequired filter

IFN-γ

Excitation 490 nm/emission 510 nm (FITC)

IL-4

Excitation 550 nm/emission 570 nm (Cy3)

5

TIPS FOR A SUCCESSFUL ASSAY
Buffers
PBS for washing and dilution should be filtered (0.2 μm) to remove any particles. We do not recommend the inclusion of Tween or other detergents in the washing and incubation buffers.

WashingPlates can be washed using a multi-channel micropipette. A regular plate washer can be used inwashing steps when sterile conditions are not required, provided that the washing head is adapted
to ELISpot/FluoroSpot plates.
SerumSerum should be selected to support cell culture and give low background staining. We recommend
the use of fetal calf serum. Alternatively, serum-free medium evaluated for cell culture can be used.

Cells
Both freshly prepared and cryopreserved cells may be used in FluoroSpot. We recommend that cryopreserved cells are rested for at least 1 hour after thawing to allow removal of cell debris before
addition to the plate. Triplicates or duplicates of 250,000 cells/well are usually recommended toassess antigen-specific responses. For polyclonal activators, the cell number should be reduced inorder to avoid confluent spot formation.

Assay controls
The number of cells responding to antigen stimulation is often compared to the number of cells
spontaneously secreting cytokine which is determined by incubating the same number of cells in
the absence of stimuli. A polyclonal activator such as anti-CD3 mAb (included in the kit) or phytohemagglutinin (1-10 μg/ml) is often included as a control for cell viability and functionality of theassay.
Co-stimulation with anti-CD28
Anti-CD28 mAb provides a co-stimulatory signal to antigen-specific responses by binding to CD28
on T cells. Addition of an anti-CD28 mAb together with antigen (step B1) can be used to enhance antigen-specific responses. However, if the concentration of anti-CD28 mAb is too high, non-specificcytokine secretion may be elevated. Anti-CD28 can be used to circumvent capture effects, which
can occur when different capture antibodies are coated in the same well, where absorption of one
cytokine may negatively affect the secretion of another cytokine. See www.mabtech.com for more
information about capture effects.
Analysis
An automated FluoroSpot reader with separate filters is required for analysis. The reader shouldbe equipped with filters for the fluorophores used. Filters should be selective for the specific wavelengths to avoid bleed-through artifacts. Spots from cells secreting multiple analytes are identified
by coordinates, i.e. based on spot position. In order to obtain accurate measurements of cells
secreting multiple analytes, the automated reader must be able to create an overlay analysis fromthe filters. To avoid fading, the plates should be kept dark and should preferably be analyzed within
1 week of development.
6

PROTOCOL SUMMARYStart
Wash x3

Cellincubation

Block/condition plate for 30 min.
Add stimuli and cells
Incubate at 37°C

Sterileconditions

Remove cells
Wash x5
Detection

Add detection antibody mix
Incubate 2h at RT
Wash x5
Add conjugate mix
Incubate 1h at RTWash x5
Add FluoroSpot enhancer
Incubate 15 min. at RT
Empty plate
Remove underdrain
and dry plate
Analysis

7

Updated on 2022/11/29
Developed and manufactured by MABTECH AB,
Sweden, whose quality management systemcomplies with the standards
ISO 9001:2015 & ISO 13485:2016.
The products are for research use only.

CERTIFIED
ISO 9001

CERTIFIED
ISO 13485

Quality management systems

Medical devices
Quality management systems

MABTECH shall not be liable for the use or handling of the product or forconsequential, special, indirect or incidental damages there from.
Mabtech AB (Head Office)SwedenTel: +46 8 716 27 00
mabtech@mabtech.com

www.mabtech.com

Mabtech, Inc.
USA
Tel: +1 513 871-4500
mabtech.usa@mabtech.com