Human Perforin ELISpot Set

ELISpot is a highly specific immunoassay method for analyzing the production of cytokines and other soluble molecules and the secretion from T cells at the single cell level, with minimal cell manipulation under conditions similar to the in vivo environment.

ELISpot is an ideal tool for studying Th1/Th2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation. Using sandwich immunoenzyme technology, LISpot can detect both secreted cytokines and single cells that produce multiple cytokines at the same time. Cytokines or soluble molecules secreted by cells are captured by coating antibodies to avoid diffusion in the supernatant, protease degradation or binding to soluble membrane receptors. After the cells are removed, the captured cytokines are displayed by the tracer antibody and the appropriate conjugate.

ELISpot (ES)

Small volumes of PRF1 ELISpot set vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

A capture antibody highly specific for the analyte of interest is coated to the wells of a PVDF bottomed 96 well microtitre plate either during kit manufacture or in the laboratory. The plate is then blocked to minimise any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any analytes produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured analyte. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using a microscope

Perforin

Human

Sandwich

Sandwich

cell suspension

Recognizes natural human Perforin

1x96 Wells

1.Pre-coated 96 well PDVF bottomed plates (5).
2.Biotinylated detection antibody (lyophilised, resuspend in 0.55ml).
3.Streptavidin-Alkaline Phosphatase conjugate (50µl).
4.Bovine Serum Albumin (BSA).
5.Ready to use BCIP/NBT substrate buffer (50ml).

1.Miscellaneous laboratory plastic and/or glass, if possible sterile.
2.Cell culture reagents.
3.Cell stimulation reagents (PMA, Ionomycin).
4.CO2 incubator.
5.Tween 20.
6.Phosphate Buffered Saline (PBS).

100 µL

1.5h

Pre-coated

1.1X Phosphate Buffered Saline (PBS) (Coating Buffer).
2.Cell culture media + 10% Serum (Blocking Buffer):For one plate add 1ml Serum (e.g. FCS) to 9ml of culture media (use same cell culture medium as used to derive the cell suspension).
3.1% BSA PBS Solution (Dilution Buffer):For one plate dissolve 0.2 g of BSA in 20 ml of 1X PBS.
4.0.05% PBS-T Solution (Wash Buffer):For one plate dissolve 50μl of Tween 20 in 100 ml of 1X PBS.
5.35% Ethanol (PVDF Membrane Activation Buffer):For one plate mix 3.5 ml of ethanol with 6.5 ml of distilled water.
6.Capture Antibody:Dilute 100μl of capture antibody in 10 mL of 1X PBS and mix well.
7.Detection Antibody:Reconstitute the lyophilised antibody with 0.55mL of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution.
8.Streptavidin - AP conjugate:Dilute in Dilution buffer according to the instructions of the supplier.

1.Add 25μl of 35% ethanol to every well.
2.Incubate plate at room temperature (RT) for 30 seconds.
3.Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper. Thoroughly wash the plate 3x with 100μl of 1X PBS per well.
4.Add 100μl of diluted capture antibody to every well.
5.Cover the plate and incubate at 4°C overnight.
6.Empty the wells as previous and wash the plate once with 100μl of 1X PBS per well.
7.Add 100μl of culture media with 10% serum to every well.
8.Cover the plate and incubate at RT for 2 hours.
9.Empty the wells as previous and thoroughly wash once with 100μl of 1X PBS per well.
10.Add 100μl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant.
11.Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours).
12.Empty the wells and remove excess solution then add 100μl of PBS-T to every well.
13.Incubate the plate at 4°C for 10 min.
14.Empty the wells as previous and wash the plate 3x with 100μl of PBS-T.
15.Add 100μl of diluted detection antibody to every well.
16.Cover the plate and incubate at RT for 1 hour 30 min.
17.Empty the wells as previous and wash the plate 3x with 100μl of PBS-T.
18.Add 100μl of diluted Streptavidin-AP conjugate to every well.
19.Cover the plate and incubate at RT.
20.Empty the wells and wash the plate 3x with 100μl of PBS-T.
21.Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper.
22.Add 100μl of ready-to-use BCIP/NBT buffer to every well.
23.Incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development.
24.Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper.

Human

AAA60065.1

Liquid

1.For research use only not to be used as a diagnostic test.
2.Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
3.Cover or cap all reagents when not in use.
4.Do not mix or interchange reagents between different lots.
5.Do not use reagents beyond the expiration date of the kit.
6.When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
7.BCIP/NBT buffer is potentially carcinogenic and should be disposed of appropriately, caution should be taken when handling this reagent, always wear gloves.

1.Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures.
2.When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels.
3.All reagents should be warmed to room temperature before use.
4.Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination.
5.Use a clean plastic container to prepare the washing solution.
6.Thoroughly mix the reagents and samples before use by agitation or swirling.

Store kit reagents between 2 and 8°C.

Immediately after use remaining reagents should be returned to cold storage (2 to 8°C). The expiry of the kit components can only be guaranteed if the components are stored properly, and if in the case of repeated use of one component, the reagent is not contaminated by the first handling.

Expiry of the kit and reagents is stated on box front labels.

Select online data sheet information is drawn from bioinformatics databases, occasionally resulting in ambiguous or non-relevant product information. It is the responsibility of the customer to review, verify, and evaluate the information to make sure it matches their requirements before purchasing the kit. Our ELISA Kit assays are dynamic research tools and sometimes they may be updated and improved.

For Research Use Only. Not for use in diagnostic procedures.

FLH2; P1; PFN1; Cytolysin; HPLH2; PFP; Lymphocyte pore-forming protein; Perforin-1

Perforin

Perforin with structural similarities to complement component C9 that is important in immunity. This protein forms membrane pores that allow the release of granzymes and subsequent cytolysis of target cells. Whether pore formation occurs in the plasma membrane of target cells or in an endosomal membrane inside target cells is subject to debate. Mutations in this gene are associated with a variety of human disease including diabetes, multiple sclerosis, lymphomas, autoimmune lymphoproliferative syndrome (ALPS), aplastic anemia, and familial hemophagocytic lymphohistiocytosis type 2 (FHL2), a rare and lethal autosomal recessive disorder of early childhood.

5551

P14222

Among its related pathways are Allograft rejection and Granzyme Pathway.

1.96-PVDF bottomed-well plates are first treated with 35% ethanol and then coated with capture antibody.
2.Incubation of cells in the coated microwell.
3.Cell removal by washing. Incubation with biotinylated antibody.
4.Incubation with streptavidin - alkaline phosphatase conjugated.
5.Addition of substrate BCIP/NBT and monitoring of spot formation.