My in-vitro culture consists of E18 rat cortical neurons. I am looking to quantify activity with calcium using GECI, RCaMP1a. The optogenetic experiment relies on the excitation of ChR2 transduced cells. Therefore I have to dual transduce my culture with both RCaMP1a and ChR2. The cell density in the plate is 150,000cm^2 in 1mL of media. The ChR2 is AAV9 and the RCaMP1a is AAV1. I usually transduce them by choosing the titer 1E10 for RCaMP and 2E10 for ChR2, and then adding them to 25uL of media and pipetting the solution directly into the wells. What I am finding is that the dual transduction wells are not as active, if at all, as the single transduction RCaMP wells. Has anyone encountered dual transduction affecting cell health and activity? If so what titer do you suggest using and is there a better way to dual transduce my cultures that would prevent this from happening? Thanks!!
Hi Madison,
Could you explain what do you mean by "... dual transduction wells are not as active"? Active in what sense?
Have you tried sequential transduction (one day, one virus)?
There is no activity in the wells that are transduced with both RCaMP1a and ChR2, meaning the cells are not firing. You can see the cells being active by the indicator RCaMP with the cells flashing red when they fire action potentials. This works great for me when I only transduce with RCaMP, however when I add ChR2 which I need for the optogenetic study, they are no longer firing APs.
I have not tried subsequent transduction. Usually when I transduce I do it on the same day at the same time to minimize shock to my cells. Do you have a suggestion for optimal dual transduction technique?
One more question regarding dual transduction (and No, I haven't done it, so cannot give a specific protocol apart from suggesting to try with one or two days separation between adding each virus) - do the cells look normal, apart from the fact that they are not active? Or maybe you see increased dying of cells, or unusual morphology?
Do you have some other virus carrying ChR2 that is not AAV9?
There is definitely a correlation between a higher titer and numerous cell death. So it seems like too much virus ultimately kills the cell. However if I decrease my titer too much I am not getting expression of RCaMP. My cell health at titers 5E9 and 1E10 looked the same-there was some cell death but overall they were healthy. Nothing unusual noted, they looked normal, just not firing APs.
I am going to try transducing them at different time points. Do you have an idea how many days I should leave between when I transduce them?
Addgene makes ChR2 carried by AAV1, which is the virus that carries RCaMP used in my experiment. I went ahead and ordered that virus to see if that makes a difference as well.
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