Hi Jens,
We have now tried this ELISPOT three times with slight changes from each, but still has not been able to see any spots. I’m attaching a photo for your reference.
This is the protocol we used for each round:
First test:
· Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate.
· pre-wet with PBS 200ul.
· blocking with RPMI1640 supplemented with 10% FCS.
· Stimulation PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.
· Cell concentrations tested: 50 000, 10 000 and 5000.
· All washing and antibody solutions according to the protocol.
· Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) (Sera Care Cat # 5420-0030(50-81-00).
· Development 30 min.
Second test:
· Surfactant-free mixed Cellulose Ester Membrane (Millipore MAHAS4510) plate.
· pre-wet with PBS 200ul.
· blocking with RPMI1640 supplemented with 10% FCS.
· Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.
· Cell concentrations tested: 50 000, 10 000 and 5000.
· All washing and antibody solutions according to the protocol.
· Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week) (Sera Care Cat # 5420-0030(50-81-00).
· Development 60 min.
Third test:
· Hydrophobic high protein binding immobile-P membrane (Millipore MSIPS4510) plates.
· pre-wet with 35% EtOH 15ul.
· blocking with RPMI1640 supplemented with 10% FCS.
· Stimulation with new batch of PMA (50ng/ml)/Ionomycin (1ug/ml) 24 hrs and media control.
· Cell concentrations tested: 100 000, 50 000 and 10 000.
· All washing and antibody solutions according to the protocol.
· Substrate: KPL BCIP/NBT Phosphatase substrate system (used in the lab previously) AND a different batch of KPL BCIP/NBT Phosphatase substrate system (which had worked in another ELISPOT that week).
· Development 60 min.
Hope you can give us some pointers of how we can make this ELISPOT work.