实验概要
The Elispot (Enzyme Linked Immuno-Spot) assay provides an effective method of measuring the antibody or cytokine production of immune cells on the single cell level. The popularity of this assay has seen resurgence in recent years as researchers attempt to gain a better understanding of immune responses in a variety of applications. The following protocol is an example of a typical Elispot assay for quantifying the number cells producing interferon-. (IFN-y) in response to antigen or non-specific activation using phytohemagglutinin (PHA). It may be optimized as necessary for other applications.
实验步骤
1. Day 1
1) Pre-wet each well with 15 μL of 35% ethanol (v/v in Milli-Q water) for one minute. Rinse with 150 μL sterile phosphate buffered saline (PBS) three times before the ethanol evaporates.
2) Caution: Ethanol volumes greater than 15 μL are not recommended. Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
3) Coat MultiScreen IP plates with 100 μL (10 μg/mL) anti-IFN-. antibody in sterile PBS (sPBS). Incubate overnight at 4°C.
2. Day 2
1) Pre-wet each well with 15 μL of 35% ethanol (v/v in Milli-Q water) for one minute. Rinse with 150 μL sterile phosphate buffered saline (PBS) three times before the ethanol evaporates.
2) Caution: Ethanol volumes greater than 15 μL are not recommended. Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
3) Coat MultiScreen IP plates with 100 μL (10 μg/mL) anti-IFN-. antibody in sterile PBS (sPBS). Incubate overnight at 4°C.
4) Pre-wet each well with 15 μL of 35% ethanol (v/v in Milli-Q water) for one minute. Rinse with 150 μL sterile phosphate buffered saline (PBS) three times before the ethanol evaporates.
5) Caution: Ethanol volumes greater than 15 μL are not recommended. Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
6) Coat MultiScreen IP plates with 100 μL (10 μg/mL) anti-IFN-. antibody in sterile PBS (sPBS). Incubate overnight at 4°C.
7) Decant blocking medium.
8) Gently plate HPBMC in 100 μL cell medium per well.
Note: To minimize over seeding of the wells, Millipore recommends adding no greater than 200,000 cells/well.
9) Incubate for 18 to 48 hours at 37°C, 5% CO2, and 95% humidity.
3. Day 3
1) Decant cells.
2) Wash plate 6 times with PBS/0.01% Tween 20. A squeeze bottle can be utilized to ensure adequate washing.
Note: If using a plate washer, increase number of wash cycles to 1.5 times the recommended number of manual cycles. It is important not to exceed 0.01% Tween 20 to prevent the possibility of leakage.
3) Dilute biotinylated anti-IFN-ã antibody to 2 μg/mL in PBS/0.5% BSA. Filter with a Steriflip-GP (Millipore, #SCGP00525) or Millex-GP (Millipore, # SLGP033RS). Add 100 μL/well.
4) Incubate for 2 hours at 37°C, 5% CO2, and 95% humidity.
5) Wash plate 6 times with PBS/0.01% Tween 20. Enzyme Conjugate and Substrate Development
6) Prepare streptavidin-alkaline phosphatase enzyme conjugate 1:1000 dilution in sPBS.
7) Add 100 μL per well of streptavidin-alkaline phosphatase. Incubate for 45 minutes at room temperature.
Note: Exceeding 1 hour incubation with enzyme conjugate will result in increased background color.
8) Decant streptavidin, wash 3 times with PBS/ 0.01% Tween 20, followed by 3 washes with PBS.
9) The final washes with PBS only are important as Tween 20 will interfere with the spot development.
10) Add 100 μL/well BCIP/NBTplus substrate. Incubate for 5 minutes.
Note: Optimization of substrate development time is critical. Development times may vary. Overdevelopment will result in increased background
11) Stop spot development under running water and wash extensively. While washing, remove underdrain and continue rinsing.
Note: To facilitate the removal of the underdrain a pair of needle nose pliers can be used. Millipore offers such a tool for this need. The part number is MSPLRS960
12) Blot plate to remove excel liquid and dry back of wells thoroughly with an absorbent wipe. This will ensure that the substrate has been completely removed from the membrane.
13) Let plate dry overnight in the dark. Spot intensity may decrease with exposure to light.
4. Day 4
1) Attach the sealing tape to the bottom of the plate and ensure that the tape sticks to the edge of each well by applying pressure to the tape with a blunt or curved implement such as the handle of a spatula.
2) Place the plate on the blue Elispot transfer pad with the membrane and tape down.
3) Insert the balck punch-out device into the well, punch out the membrane by applying light pressure. A faint click sound indicates that the membrane has transferred to the sealing tape. Repeat for all wells.
4) Total time for removing all membranes is approximately 2-3 min.
5) Store tape in a plastic sheet protector along with protocol and evaluation results.
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