M94xx pelispot pair.doc

PeliSPOT Pair
370 tests (>3 plates)
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INTENDED USE
This antibody pair is suitable to perform an ELISPOT assay for the quantification of the frequency of human cells secreting the protein of interest. CONTENTS
This product contains sufficient material for 370 tests. PRECAUTIONS FOR USE
1. PeliSPOT Pair is intended for research purposes only, ingredients are not for in 2. Handle all blood, tissue and cell samples with care to prevent transmission of 3. Sodium azide inactivates HRP, do not use sodium azide containing solutions 4. Do not use PBS tablets for coating. 5. Centrifuge all vials before use (1 minute at 3,000 x g). 6. The supplied reagents have been tested and approved in combination with the 7. Aseptic Procedures; Use sterile buffers and aseptic conditions; use laminar
flow hood for procedures.
8. The supplied reagents have only been tested and approved for use on MSIPS45 microplates from Millipore. Make sure not to scrape the bottom of the wells and do not let the membrane dry out. * in combination with a single cell culture tray, Millipore MAMC S01 CELL PREPARATIONS AND STIMULI
Freshly isolated or frozen peripheral blood mononuclear cells (PBMC) as well as cell lines or cultured cells can be used. After being drawn, anti-coagulated blood is kept at room temperature (18 to 25 °C) for a maximum of 24 hours. PBMCs are isolated from venous blood by density centrifugation according to the manufacturer's protocol. Wash the cells twice with cell culture medium. 1301061117

Add a mitogen as a positive secretion control, for example PMA (1 ng/ml) and
Ionomycin (0.5 µg/ml). Wells that exceed the number of 2.5×104 mitogen-
stimulated cells per well are generally not countable due to high protein
secretion.
The optimal concentration of the antigen during antigen-specific stimulation with
e.g. peptides and the optimal number of cells per well depend on the cell type
and excretion marker and, therefore, they have to be determined by the
investigator. Start with a maximal cell number of 2×105 cells per well and an
antigen concentration between 1 and 10 µg/ml.
V.
RECOMMENDED REAGENTS AND BUFFERS*
: 10 mM Phosphate buffered saline, pH 7.2-7.4 : IMDM or RPMI-1640 with 5% FCS, 2 mM Glutamine, 100 U/ml Penicillin, 100 µg/ ml Streptomycin : PBS + 0.005% Tween-20; 0.2 µm filtered * For aseptic conditions these components should always be used sterile
Available at Sanquin Reagents:
order number

Dilution buffer
GENERAL ASSAY PROTOCOL
This is a suggestion only, optimal conditions should be determined
experimentally.
Check the included specification sheet for cytokine dependent modifications

Day 1 Aseptic Procedures
1. Activate the PVDF membrane of the microplate with 70% ethanol for 60
seconds 15 µl for MSIP plates and for MAIP plates: 100 µl 96% ethanol for 10 minutes at room temperature. 2. Rinse once with distilled water and wash once with PBS. 3. Dilute the coating antibody 1:100 in PBS and add 50 µl per well. 1301061117

4. Incubate overnight at 2 to 8°C.
Day 2 Aseptic Procedures
5. Wash the microplate five times with wash buffer.
6. Add the cell suspension and cell stimulus of interest.
7. Incubate 16 – 44 hours at 37°C in a CO2 incubator.
During this period make sure that the microplate is completely horizontal and
do not agitate or move the microplate.

Day 3 Non aseptic Procedures
8. Empty the wells, rinse underside of the membrane with distilled water, wash the microplate five times with wash buffer and tap microplate on absorbent paper. 9. Dilute the biotinylated antibody 1:100 in dilution buffer and add 100 µl per 10. Incubate for one hour at room temperature. 11. Empty wells, rinse underside of the membrane with distilled water, wash the microplate five times with wash buffer and tap microplate on absorbent paper. 12. Dilute the streptavidin-poly-HRP 1:6,000 in dilution buffer and add 100 µl 13. Incubate for one hour at room temperature. 14. Empty wells, rinse underside of the membrane with distilled water, wash the microplate five times with wash buffer and tap microplate on absorbent paper. 15. Add 50 µl substrate per well, incubate for 4-15 minutes at room temperature. Monitor spot intensity by eye. 16. Rinse underside of the membrane and wash the microplate with an excess 17. Dry the microplate and count the number of spots preferably using the Please inquire for information or demos of the A.EL.VIS spot analyzer
systems
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Source: http://www.immunologia.biz/eliodcz/m94xx.pdf