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Membrane is grey and there is a lot of background in some wells

Guest Anita2023-11-13T07:30:25Z

Hi,

I have run my first fluorospot and would like help troubleshooting why some wells have some odd background/low staining. The membrane also appears to have a grey colour like it is not dry properly but I completed the assay 3 days ago. It is the IFN-gamma, granzyme B, TNF-alpha kit.

My samples are human naive CD8 T cells that were stimulated for different timepoints of 0 days up to 14 days before washing with RPMI media and then adding cells to the fluorospot plate. The cells were incubated on the plate for 20 hours with stimulation media. There is no pattern in stimulation time with the wells that have this background and all wells were stimulated with the same cocktail of Anti-CD3, Anti-CD28, Anti-CD2 and IL-2.

I have attached a powerpoint file with photos of the plate showing the membrane and examples of a good well and well with odd background. Please let me know your thoughts. Thanks!

Fluorospot troublshooting.pptx

Christian@mabtech.com2023-11-15T13:45:12Z

Admin

Is this a precoated FluoroSpot plate, or have you coated yourself (ie FluoroSpot Flex)?

The wells where the assay has worked look great. The "wet" looking wells look bad.

Could there that you have added too much fluorscence enhancer? This can cause a "wet" effect which can last long, even 3 days.

But at the same time, this looks to me like dried out phenomenon. My suspcision is that this is a FluoroSpot Flex kit and that the membrane has been allowed to dry out in certain wells during either coating, experiment setup or during detection. Air exposure to the membrane should be minimized, especially for fresh coated plates.

best,

Christian

Guest Anita2023-11-16T06:36:43Z

Hi Christian,

Thanks for the reply. No this was a pre-coated plate from the kit (https://www.mabtech.com/products/fluorospot-plus-human-ifn-ggranzyme-btnf-fsp-013609). I added the same fluorescence enhancer to all the wells but if itcan cause a wet effect, maybe I didn't tap it off enough before leaving to dry. Could this be the cause?

Thanks,

Anita

Guest Anita2023-11-16T22:28:58Z

On 11/16/2023 at 12:45 AM, Christian@mabtech.com said:

Is this a precoated FluoroSpot plate, or have you coated yourself (ie FluoroSpot Flex)?

The wells where the assay has worked look great. The "wet" looking wells look bad.

Could there that you have added too much fluorscence enhancer? This can cause a "wet" effect which can last long, even 3 days.

But at the same time, this looks to me like dried out phenomenon. My suspcision is that this is a FluoroSpot Flex kit and that the membrane has been allowed to dry out in certain wells during either coating, experiment setup or during detection. Air exposure to the membrane should be minimized, especially for fresh coated plates.

best,

Christian

@Christian@mabtech.com Please see my reply above.

Kind regards,

Anita

Christian@mabtech.com2023-11-17T12:00:15Z

Admin

On 11/16/2023 at 7:36 AM, Guest Anita said:

Hi Christian,

Thanks for the reply. No this was a pre-coated plate from the kit (https://www.mabtech.com/products/fluorospot-plus-human-ifn-ggranzyme-btnf-fsp-013609). I added the same fluorescence enhancer to all the wells but if itcan cause a wet effect, maybe I didn't tap it off enough before leaving to dry. Could this be the cause?

Thanks,

Anita

Yes too much Fluorscence enhancer can lead to problems, but it feels unlikely in this situation. Why just a few wells?

After you remove the plastic underdrain, the membrane is totally exposed from the backside. Re-wetting of the membrane from the backside can cause very strange effects. Especially if the liquid is some sort of detergent or similar. Rather easy to leave the plate in a small pool of detergent on the lab-bench. One has to be very careful after removal of underdrain.

Try drying the plate so the wetting effect disappears. If this is not possible then it must be something else than FS enhancer.

Guest Anita2023-12-04T23:00:22Z

On 11/17/2023 at 11:00 PM, Christian@mabtech.com said:

Yes too much Fluorscence enhancer can lead to problems, but it feels unlikely in this situation. Why just a few wells?

After you remove the plastic underdrain, the membrane is totally exposed from the backside. Re-wetting of the membrane from the backside can cause very strange effects. Especially if the liquid is some sort of detergent or similar. Rather easy to leave the plate in a small pool of detergent on the lab-bench. One has to be very careful after removal of underdrain.

Try drying the plate so the wetting effect disappears. If this is not possible then it must be something else than FS enhancer.

Hi @Christian@mabtech.com

The plate had dried when those images were taken but it took a long time to dry. Maybe FS enhancer residue? But as you said, it is odd that it is not all wells.

Could this occur if there are too many dead cells? Could cell debris cause an effect like this?

Thanks,

Anita

Christian@mabtech.com2023-12-06T08:15:04Z

Admin

On 12/5/2023 at 12:00 AM, Guest Anita said:

Hi @Christian@mabtech.com

The plate had dried when those images were taken but it took a long time to dry. Maybe FS enhancer residue? But as you said, it is odd that it is not all wells.

Could this occur if there are too many dead cells? Could cell debris cause an effect like this?

Thanks,

Anita

Could be enhancer issue, especially if some of it ended up on the backside of the membrane.

I have a hard time seeing how more dead cells could cause this effect, but I know that certain peptides can cause very strange outcomes. Some will bind strangely to the PVDF and cause strange issues. Also, very high concentrations of DMSO can induce super strange artefacts. What was the stimulation in these particular wells?

Guest anita2023-12-11T02:17:22Z

On 12/6/2023 at 7:15 PM, Christian@mabtech.com said:

Could be enhancer issue, especially if some of it ended up on the backside of the membrane.

I have a hard time seeing how more dead cells could cause this effect, but I know that certain peptides can cause very strange outcomes. Some will bind strangely to the PVDF and cause strange issues. Also, very high concentrations of DMSO can induce super strange artefacts. What was the stimulation in these particular wells?

@Christian@mabtech.comAll wells had the same pre-stimulation which was CD3, CD28 and IL-2 before adding cells t the fluorospot plate but many of the wells with the effect were stimulated for longer period of time, up to 2 weeks.

Christian@mabtech.com2023-12-15T11:43:22Z

Admin

After leaving the plate for a few days, have the "wet" looking wells totally dried out and become white?