Description
Standard ELISPOT Protocol Buffers, Media, and Other Reagents for ELISPOT Assays Coating Buffer: Dulbecco's Phosphate-Buffered Saline (PBS): 8 g NaCl; 0.2 g KCl; 1.44 g Na2HPO4•7H2O; 0.24 g KH2PO4; add dH2O to 1 liter. Adjust pH to 7.2, autoclave or sterile filter (0.2 μm-pore) and store at 4°C. Complete Tissue Culture Medium A medium consisting of RPMI 1640 (Bio-Whittaker, Cat. No. 12-167Q) or another suitable medium containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin-L-Glutamine (Gibco-BRL Cat. No. 10378-016), and 5 × 10-5 M 2-mercaptoethanol is often used for culturing human, non-human primate, and rodent cells. PBS-Tween®: PBS containing 0.05% Tween®-20 (Sigma, P-1379; 0.5 ml Tween®-20 per 1 L PBS). Dilution Buffer: PBS containing 10% FBS. Substrate Solution: Utilize the BD™ ELISPOT AEC Substrate Set (Cat. No. 551951) for convenience from BD Biosciences. Protocol 1. Coating Antibody: a. Dilute the Capture Antibody to the recommended concentration with Coating Buffer. Add 100 μl of diluted antibody solution to each well of an ELISPOT plate. b. Replace the ELISPOT plate lid and store plates at 4°C overnight. 2. Blocking: a. Discard the Coating Antibody. Wash the wells 1 time with 200 μl/well of Complete Tissue Culture Medium that contains 10% fetal bovine serum. b. Add 200 μl/well of complete tissue culture medium; replace the ELISPOT plate lid and allow blocking for 2 hours at room temperature. 3. Cell Activation: Specific activation protocols including cell concentrations and incubation times will vary depending on the cell type, choice of stimulus, and target analyte of interest. a. Discard the Complete Tissue Culture Medium. b. Prepare mitogen, antigen, or other biological agent diluted in Complete Tissue Culture Medium for treating cells. Add 100 μl/well to ELISPOT plate. c. Prepare cell suspensions at different densities, (eg, ranging from 1 × 10^5 cells/ml to 2 × 10^6 cells/ml). Note that appropriate negative controls should be prepared by adding cells to wells without a particular treatment agent and by establishing background wells without cells, (ie, wells that just receive the Complete Tissue Culture Media). Cell titrations can be performed either in another cell culture plate or in tubes and then transferred to the ELISPOT plate microwells or performed directly in the ELISPOT plate. Care should be taken not to touch or damage the coated microwell surface. Cells should be added in 100 μl volumes to ELISPOT plate microwells. d. After adding the cells, replace the ELISPOT plate lid and incubate the plate at 37°C, 5% CO2 and 99% humidity. The duration of the incubation time will vary depending on the agent used to treat cells to secrete (or not secrete) the analyte of interest (eg, cultures are usually established for 2-48 hr). 4. Detection Antibody: a. Aspirate the cultured cell suspensions from the ELISPOT plate microwells. After step 3, aseptic conditions are not required. Wash and soak the wells 2 times with 200 μl/well of distilled water (dH2O). Allow wells to soak for 3-4 min at each wash step. b. Wash wells 3 times with 200 μl of PBS-Tween® per well. Discard Wash Buffer. c. Dilute biotinylated Detection antibody in Dilution Buffer. Add 100 μl per well. d. Replace the ELISPOT plate lid and incubate for 2 hr at room temperature. 5. Streptavidin-Horseradish Peroxidase (Streptavidin-HRP) [Cat. No. 557630]: a. Discard Detection antibody solution. Wash wells 3 times with 200 μl/well of PBS-Tween®. Allow wells to soak for 1-2 min at each wash step. b. Dilute Streptavidin-HRP in Dilution Buffer. Add 100 μl of diluted Streptavidin HRP per well. c. Replace the ELISPOT plate lid; incubate for 1 hr at room temperature. 6. Substrate: a. Discard Streptavidin HRP solution. Wash wells 4 times with 200 μl of PBS-Tween® per well. Allow wells to soak for 1-2 min at each wash step. b. Wash and soak wells 2 times with 200 μl of PBS per well. c. Add 100 μl of AEC Substrate Solution prepared using the BD™ ELISPOT AEC Substrate Set (Cat. No. 551951) to each well. Monitor spot development at RT from 5-60 min. Do not let color overdevelop. This will lead to high background. d. Stop the substrate reaction by rinsing wells thoroughly with dH2O. e. Air-dry plate for 2 hr or overnight in the dark until the plate is completely dried. Removal of plastic tray under 96-well plate facilitates drying. Store the plate in a sealed plastic bag, in the dark, prior to analysis. f. Enumerate brownish spots manually by inspection under a dissecting microscope (or stationary magnifying glass) or automatically using an ELISPOT analyzer and software. Warning: Streptavidin-HRP (component 51-9000209) contains 0.004% of a CMIT/MIT mixture (3:1), which is a mixture of: 5-chloro-2-methyl-4-isothiazolin-3-one [EC No 247-500-7] and 2-methyl-4-isothiazolin-3-one [EC No 220-239-6] (3:1). Hazard statements May cause an allergic skin reaction. Precautionary statements Avoid breathing dust/fume/gas/mist/vapours/spray. Wear protective gloves/protective clothing/eye protection/face protection. Contaminated work clothing should not be allowed out of the workplace. If skin irritation or rash occurs: Get medical advice/attention. IF ON SKIN: Wash with plenty of water. Dispose of contents/container in accordance with local/regional/national/international regulations.
Streptavidin was conjugated with the enzyme under optimal conditions. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
4. Hazardous Ingredient: ProClin™ 150. Avoid exposure to skin and eyes and ingestion. Wash exposed skin with soap and water. Flush eyes with water.
5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
6. ProClin is a trademark of Rohm and Haas Company.
7. For U.S. patents that may apply, see bd.com/patents.
