Missing cells from immunopanning?

Question

Why do I not have any cells left in my positive panning plate after transferring from the negative panning plate during immunopanning? I am trying to purify retinal ganglion cells from postnatal rats and use a Thy1.1 antibody harvested from a hybridoma cell line supernatant in the positive panning plate. Following transfer of the cell suspension from the negative to the positive panning plate there are no cells in the pre-trypsinization supernatant, nothing stuck to the panning plate, and no cells in the post-trypsinization supernatant so I have no idea where they could have gone. Checking the negative panning plates however shows that there are many cells stuck to those plates, so could my RGCs have been lost at some point during the transfer and when?Thanks v much in advance for your help.

Answer 1

It sounds to me that you are most likely losing your retinal ganglion cells (RGCs) during the negative panning step. Since you are seeing many cells retained on the negative panning plates, it is possible that RGCs are sticking there due to overly strong binding or non-specific interactions. This can happen if blocking is insufficient, incubation times are too long, or the negative selection antibodies have high cross-reactivity.

Here are a few points to keep in mind:

Blocking and incubation times — Over-incubation on the negative plates can result in a loss of target RGCs. Ensure that you are optimizing your blocking reagent (typically BSA or serum), using freshly prepared buffers, and limiting incubation times.

Antibody efficiency on the positive plate — You are using Thy1.1 hybridoma supernatant, which can vary in antibody concentration. If the positive panning plate is not coated well, RGCs may fail to adhere and could be lost during washes. Make sure you are incubating the plates long enough (typically overnight at 4°C), using sufficient antibody concentration, and properly washing and blocking the plates before use.

Transfer loss and handling — RGCs are extremely fragile, especially from postnatal rats. Ensure that transfers between plates are done very carefully, with minimal time delays and using proper buffers (typically add DNase to avoid clumping).

Washing the positive plate — Washes should be gentle; overly vigorous washing can dislodge weakly bound RGCs.

Control test — If you haven't already, try skipping the negative panning and plating directly onto the positive panning plate. If you still recover few or no cells, this suggests the issue is more related to the positive plate or antibody coating.

To summarize, based on your description, the most likely step where you're losing RGCs is at the negative panning plate. I would first optimize that step with shorter incubation, proper blocking, and then confirm that your positive plate is efficiently coated and working. Good luck! Purifying RGCs can be tricky, but after optimizing your process the yield will improve dramatically.