小鼠干扰素-γELISpot试剂盒(ALP)(板条可拆卸)

Intended Use

The kit applies for the detection of mouse IFN-γ at the cellular level by counting IFN-γ-secreting cells.

PRINCIPLE OF THE ASSAY

The enzyme-linked immunospot (ELISPOT) assay employs a sandwich ELISA-based methodology. A 96-well polyvinylidene difluoride (PVDF) membrane plate, pre-coated with mouse IFN-γ-specific monoclonal antibodies, captures mouse IFN-γ secreted by activated cells. Following cell removal and washing, biotin-conjugated secondary antibodies targeting IFN-γ are added to form antibody-antigen-antibody "sandwich" complexes. Subsequent incubation with streptavidin-alkaline phosphatase (ALP) conjugates enables biotin-mediated binding. Addition of chromogenic substrate culminates in the formation of insoluble spots at the membrane interface, with each spot representing an individual IFN-γ-secreting cell. Spot quantification is achieved by automated ELISPOT analysis or manual counting using a microscope.

KIT CONTENTS AND STORAGE

Please use the kit before the expiration date.

Components	Amount	Preparation instructions	storage
Pre-coated microplate	1plate (96 tests)	Once removed from vacuum bag, the plate should be used as soon as possible.	Vacuum storage can store at 2-8℃ until expiration date.
Detection Antibody	1 vial	Dilute the detection antibody 1:350 in PBS containing 0.5% fetal bovine serum (PBS-0.5% FBS) before use. Dilute fresh as needed.	Primary liquid are stable at 2 - 8℃ until expiration date. To be reconstituted, the working fluid is used within the working day and discard. So dilute fresh as needed.
Streptavidin-ALP	1 vial	Dilute the Streptavidin-ALP 1:170 in PBS-0.5% FBS.
BCIP/NBT-plus Dilution	1 bottle	BCIP/NBT-plus Solution A and the Dilution should be mixed together in a 1:250 volume, followed by the addition of Solution B in the same volume as Solution Aand mixed gently.The volume ratio of Solution A:Dilution:Solution B is 1:250:1. For example: pipette 40 µl of Solution A into 10 ml of the Dilution buffer, mix thoroughly, then add 40 µl of Solution B and mix gently. Dilute fresh as needed.
BCIP/NBT-plus Solution A	1 bottle
BCIP/NBT-plus Solution B	1 bottle

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 bovine serum (FBS) asused 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: Fewer cells such as 1×10⁵ cells/wellare needed for polyclonal activators, e.g., ConA (0.25-2.5 µg/ml).
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 setduplicate 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 ofthe detection antibody andStreptavidin-ALP. The PBS should be filtered (0.2 μm) 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 detection antibody 1:350 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 1hour 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 andadd 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 theunderdrain (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 a temperature 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