Directions for washing ELISPOT and FluoroSpot plates

Directions for washing ELISPOT and FluoroSpot plates

Directions for washing of PVDF membrane-bottomed plates
For effective washing of PVDF membranes, a squirt bottle with a wide spout has shown to achieve the best results. The bottle should be used to thoroughly flush all emptied wells of the plate with wash buffer. While flushing, the wells are completely filled with wash buffer and subsequently emptied by a firm ‘shake-out’ action. Repeat this as many times as mentioned in the manual. After washing, the plate is emptied by tapping it upside down on absorbent tissue paper. 
Additional washing of the backside of the PVDF membrane is needed after the incubation steps with detection antibody and conjugate to further reduce background staining and to prevent blank areas in between the spots. Therefore, remove and discard the plastic underdrain of the plate and use the squirt bottle to wash the backside of the membrane with wash buffer. After washing, the wash buffer is removed by a gentle ‘shake-out’ action.

Notes:

• All washing steps must be performed with freshly prepared wash buffer (see manual).
• Do not puncture the PVDF membrane by pipetting/washing procedures. The membrane is fragile and may easily be damaged. 
• During incubation with blocking solution, membrane-leakage occasionally occurs. However, this phenomenon does not negatively affect assay results. 

To T cell ELISPOT assay guidelines, B cell ELISPOT assay guidelines or to FluoroSpot assay guidelines