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How can I wash adherent cells completely?

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Dear Mabtech,

Recently, I bought IFN-g ELISPOT kit pro and tried to find condition. I used PBMC and adherent cells, after 24 hours, I did assays on manufactured protocol. But I couldn’t wash out cells completely and I couldn’t detect signals (positive control is working). Then, I’m thinking to use Tween-PBS for the wash buffer, but your company doesn’t recommend it, I know. How can I solve this problem? Can I use accutase or Trypsine enzyme for a while?

I am attaching images from the wells.

 Adherent_cells.pptx

Regards,

T

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Hi T,

I think one should avoid using trypsin or accutase as they are both proteolytic enzymes and can lead to the digestion of both the antibodies and the analyte. Instead, we recommend a wash protocol including EDTA. In short, adherent cells are removed by first washing 3x with PBS, and then a 10 minute incubation at 37°C with 100ul PBS supplemented with 1mM EDTA in each well. After that, you just wash once again with PBS, and the membranes should be clean from cells. Please find the steps below and attached as a pdf.

Mabtech_ELISpot_adherent_cells_protocol.pdf

 

1. Wash the cells 3x with 200ul PBS. If the cells should be recovered, this step should be done sterile in a sterile hood. If the cells will be discarded. The washes can be

done in an automatic washer.

NB! Wash vigorously to make sure that all medium (containing Ca2+ and Mg2+) is washed away before adding the 1 mM EDTA in step 2.

 

2. Add 100 ul PBS supplemented with 1mM EDTA. If the cells should be recovered, this step should be performed sterile. Incubate at 37°C for 10 minutes.

NB! Since EDTA is more effective at 37°C it should ideally be pre-warmed before being added to the plate. For difficult cells, the time of incubation could be increased to 15 minutes. For further effect, you could also tap the side of the plate with your hand or put the plate on a lab shaker.

 

3. If the cells are to be recovered, hit the plate against the palm of your hand a few times and re-suspend vigorously with a pipette. Remove the cells to another plate and continue with your ELISpot/FluoroSpot assay. If the cells are not to be recovered, wash them away using 1x 200 ul PBS and then continue with your ELISpot/FluoroSpot assay as usual.

NB! Do not touch the membrane with the pipette tips!

 

Kind regards,

Jens

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