rev: March 16, 2005
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PeliSPOT (tm) Human ELISPOT Reagent Set
-manufactured by CLB Reagents
cat#RDI-M2536clb Human Granzyme B $750.00
cat#RDI-M2533clb Human IFN-gamma $500.00
cat#RDI-M2514clb Human IL-4 $500.00
Cytokine Pelispot Antibody pairs NEW $375.00/set
cat#RDI-M9433clb Human IFN-gamma
cat#RDI-M9414clb Human IL-4
cat#RDI-M9410clb Human Il-10
cat#RDI-M9413clb Human IL-13
cat#RDI-M9515clb MOUSE IL-5
-see also new dedicated reagent sets for Elispot (PeliSpot)
-for BIO-Plex(tm) Luminex (tm) system see-
Jager et al, "Similtaneous Detection of 15 human Cytokines in a Single Sample of Stimulated Peripheral Blood Mononuclear Cells", Clinical and Diagnostic Laboratory Immunology, Jan 2003, p133-139
plates per kit
* Peilpair antibody dilutions (optimized dilution should be determined individually)
-see also Granzyme B Peli-Spot(tm kit) for Granzyme B
-see also IFN-Gamma Elispot kit
-Coat ( e.g. ethanol activated PVDF plates ) with catching antibody* to cytokine; 50 Ál/well in coating buffer overnight at room temperature (RT).
-Wash the plate 4x with PBS; 1x with IMDM or with RPMI 1640.
-Incubate with blocking medium; 100 ul/well 1 h at 37oC.
-Empty plate and fill with cells (titration) and stimuli.
-Incubate at 37 oC in CO2 incubator. Incubation time depends on stimulus and cytokine under study.
-Wash the plate 5x with washing buffer.
-Add biotinylated antibody* to cytokine 100 ul/well in HPE and incubate 1h at RT on a shaker.
-Wash 5x with washing buffer.
-Incubate for 5min at RT with 100 Ál streptavidin-poly HRP (1/2000 in PBS + 2% milk) on shaker.
-Wash 5 x with washing buffer.
-Stain for ~10 minutes at RT with 3-amino-9-ethylcarbazol.
-Wash with H2O and dry.
Coating buffer: 0.1 M NaHCO3, pH 9.5
PBS 10 mM sodium phosphate 0.14 M NaCl pH 7.2
Culture medium RPMI 1640 or IMDM with 5 % FCS, pen/strep,
50 µM 2-mercaptoethanol; human transferin 20 µg/ml.
Blocking medium RPMI or IMDM containing 10 % FCS, pen/strep and 50 M 2-mercaptoethanol
Washing buffer PBS + 0.02 % Tween-20 (PT).
HPE Elisa High Performance buffer produced by CLB
3-amino-9-ethylcarbazol stock solution 10 mg/ml in dimethylformamide; For use in this assay dilute 1:30 in 50 mM Na acetate pH 5.0 and filter through a 0.45 µM filter. Then add 0.015 % H2O2.
To measure T cell cytokines (IL-2, IL-4, IL-13, IFN, TNFá and GM-CSF) the following stimuli can be used:
- Ca-ionophore (1 µM ionomycine) + PMA 1ng/ml. Stimulation overnight or
- anti-CD3 1 µg/ml + anti-CD28 1 µg/ml. Stimulation is for 2,3 or 4 days.
To measure monokine production (IL-1, IL-6, IL-8, IL-10, IL-12 and TNFá) stimulation can be carried out with LPS 1ng/ml or with SAC (1:1000).
For a number of monokines the plates are stimulatory by themselves.
-see some combinations under the individual cytokine antibodies which can be accessed through the cytokine table
RDI Divison of Fitzgerald Industries Intl
34 Junction Square Drive
Concord MA 01742-3049
phone (978) 371-6446 or (800) 370-2222
fax (978) 371-2266
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for recombinant cytokines and antibodies , click here
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