Human IL-2 ELISpot Kit (ALP)

ASSAY PROCEDURE

1. Plate Set-up (sterile conditions)
Remove the plate from the sealed package and wash 4 times with sterile PBS (200 µl/well).

Condition the plate with sterile RPMI-1640 culture media (200 µl/well) containing 10% fetal calf serum (FBS) as used for the cell suspensions, Incubate for at least 30 minutes at room temperature.

2. Incubation of cells in plate (sterile conditions)
Empty the plate, then add stimulus followed by the 100 µl cell suspensions to each well. The concentration of cell and stimulus should be adjusted according to the actual situation. The recommended group settings are as follows.
a.Positive control: The anti-CD3 mAb, included in the kit, is recommended as a positive control at a dilution of 1:125. Add 10 µl of the stimulus per well. Fewer cells such as 1.5×10⁵cells/well are needed for this positive control group.
b. Negative control: The cell concentration is kept consistent with the experimental wells in the absence of stimulus.
c. Background control: No cells, no positive stimulus, add to the medium.
d. Experimental group: The recommended cell number per well is 1.5–2.5 × 10⁵, and add your own stimulus as required by the experiment (prepare the stimulus using culture medium).

Note: It is recommended to set duplicate wells for samples to be tested.

Put the plate in a 37℃ and 5% CO₂ incubator for 18-48 hours.Do not move the plate during this period and it is recommended to wrap the plate in aluminum foil to avoid evaporation.

3. Detection of spots

Note: Use PBS containing 0.5% FBS (PBS-0.5% FBS) for dilution of the Biotin-conjugated antibody antibody and Streptavidin-ALP. The PBS should be filtered (0.2 um) for optimal results.Avoid the inclusion of Tween or other detergents in the washing and incubation buffers.

Remove the cells by emptying the plate and wash 5 times with PBS, 200 µl/well.

Note: Soak in the PBS for 1 minute to ensure thorough cleaning. The same requirement applies to all subsequent washing steps.

Dilute the Biotin-conjugated antibody 1:370 in PBS-0.5% FBS. Add 100 µl/well and incubate for 2 hours at room temperature.
Note: Diluted detection antibody can be filtered (0.2 µm) to reduce the risk of unspecific background.

Empty the plate and wash 5 times with PBS, 200 µl/well. Dilute the Streptavidin-ALP 1:170 in PBS-0.5% FBS and add 100 µl/well. Incubate for 1 hour at room temperature. Empty the plate and wash 5 times with PBS, 200 µl/well. Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45 µm filter and add 100 µl/well. Develop until distinct spots emerge in positive wells (usually 5-30 minutes).

Note: Too long the color development time will cause the background color in the experimental well to become darker.

Stop color development by washing extensively in tap water. If desirable, remove the underdrain (the soft plastic under the plate) and rinse the underside of the membrane.
Leave the plate to dry. Plates should be completely dry before analysis. Inspect and count spots in an ELISpot reader or in a microscope.

Note：Do not dry the microplate at atemperature higher than 37°C; this may cause cracking of the membrane filters.

For the option to re-analyze later, store plate in the dark at room temperature.

TROUBLESHOOTING GUIDE

Problem	Possible reason	Solution
Dark background of the PVDF membrane	*Plate is insufficiently washed * The membrane is wet	*Increase the washing times to ensure soaking time *Microplates should be analyzeduntil the PVDF membranes are completely dry
Spots appear in the negative control wells	*Endotoxins or other contaminants in the medium, serum, or DMSO may activate immune cells *The cells are contaminated *Cell viability is low	*Use endotoxin-free reagents *Pay attention to aseptic procedures in the step of Plate Set-up and Incubation of cells in plate *Ensure high viability (recommended viability > 90%) as well as functionality of the cell sample
Spots are irregular in shape and severely clumped	*Cell disruption *Cell aggregated	*Optimize the cell separation process to ensure cell activity *The cell suspension is thoroughly mixed toensure a unicellular state
Positive Control spots were low and fuzzy	*Enzyme activity is decreased *The temperature of the Substrate reagent is low and oxidized *The titer of the stimulus is low	*Increase enzyme concentration *Balance the color reagent to room temperature before use, do not use reagents that have oxidized precipitation *Increase the concentration of stimulus
The spots are unevenly positioned and have an edge effect	*Shake the cell plate after plating, causing cell displacement *Add the stimulus too vigorously to flush the cell suspension *Stack the plates in the incubator	*After plating, try not to shake the plate, and gently pan it into the incubator *Use the method of adding the stimulus first and then the cells *Place each plate individually on the shelf to allow an even distribution of heat to each microwell
Not all spots are counted in the crowded well despite algorithm and parameter adjustments	*Spots are confluent. Single spots cannot be discriminated	*Decrease the number of cells added per well