Components
Fas Ligand Biotinylated detection antibody
Clone B-B34 Capture antibody
Material not included
96 PVDF-bottomed-well plates.
Cell culture media.
CO2 incubator.
70% ethanol.
Tween 20.
Phosphate buffered saline.
Streptavidin - Alkaline Phosphatase conjugated.
Bovine Serum albumin.
Dry skimmed milk : non sterile Elispot / Liquid sterile milk: sterile Elispot.
Substrate Solution (BCIP/NBT).
Reagent Preparation
1.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.
2.Streptavidin alkaline phospatase:Dilute in PBS 1% BSA.
3.Phosphate buffered saline (10X Concentrate solution):For 1 liter weigh: 80g NaCl; 2g KH2PO4; 14.4g Na2HPO42H2O. Add distilled water to 1 liter. Check that pH is 7.4 +/- 0.1. Dilute the solution to 1X before use.
4.2% dry skimmed milk in PBS:For one plate dissolve 0.2 g of powder in 10 mL of 1X diluted PBS.
5.1% BSA in PBS.
6.0.1% Tween in PBS.
7.70% ethanol in water.
Assay Procedure
1.Incubate PVDF-bottomed-well plates with 25µl / well of 70% ethanol for 30 sec at room temperature.
2.Empty wells and wash three times with 100µl / well of PBS.
3.Pipette 100µl of Fas Ligand capture antibody in 10 mL of PBS. Mix and dispense 100 µl into each well, cover the plate and incubate overnight at 4°C.
4.Empty wells and wash once with 100 µl of PBS.
5.Dispense 100 µl of 2% skimmed dry milk in PBS into wells, cover and incubate for 2 hours at room temperature.
6.Empty wells by flicking the plate over a sink and tapping it on absorbent paper.
7.Wash plate once with PBS.
8.Dispense into wells 100 µl of cell suspension containing the appropriate number of cells and appropriate concentration of stimulator. Cells may have been previously in-vitro stimulated (Indirect ELISPOT). Cover the plate with a standard 96-well plate plastic lid and incubate cells at 37°C in a CO2 incubator for an appropriate length of time (10-15 hours).
9.Empty wells by flicking the plate over a sink and gently tapping it on absorbent paper.
10.Distribute 100µl of PBS-0.1% tween 20 in wells and let sit for 10 min at +4°C.
11.Wash wells three times with PBS-0.1% tween 20.
12.For 1 plate dilute 100µl of reconstituted Fas Ligand detection antibody into 10 mL of PBS containing 1% BSA. Distribute 100µl in wells, cover the plate and incubate 1 hour 30 min at +37°C.
13.Empty wells and wash three times with PBS-0.1% tween 20.
14.Distribute 100µl of the streptavidin-Alkaline phosphatase conjugate dilution into wells. Seal the plate and incubate for 1 hour at +37°C.
15.Empty wells and wash three times with PBS-0.1% tween 20. Remove all residual buffer by repeated tapping on absorbent paper.
16.Distribute 100µl of ready-to-use BCIP/NBT buffer in wells.
17.Let the reaction go for about 2-10 min at room temperature. Monitor spot formation visually.
18.Rinse wells three times with distilled water.
19.Dry wells. Read spots. Note that spots may become sharper after one night at +4°C. Store the plate at room temperature away from direct light.
Precaution of Use
All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance.
Handling Advice
1.Kit components should be stored as indicated. All the reagents should be equilibrated to room temperature before use.
2.Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid crosscontamination; for the dispensing of the substrate solution, avoid pipettes with metal parts.
3.Thoroughly mix the reagents and samples before use by agitation or swirling.
Storage
Store kit at 2-8°C immediately upon receipt.
Note
Spots may become sharper after overnight incubation at 4 oC. Plate should be stored at room temperature away from direct light, but color may fade over prolonged periods so read results within 24 hours.
Restrictions
For Research Grade Use Only.