Team:Penn/Notebook

09/20/2011 (Avin, Mike M, Peter, Anthony)

First time doing calcium imaging. Brought over 293T's in a 6 well plate, which had been transfected with CatCh. Also had positive control cells on which we used ionophores. Used the Meaney Lab confocal and x-rhod1 dye. Media had 2mM Calcium. 1uM Ionomycin addition did not change x-rhod1 fluorescent intensity in Anthony's neurons.

Also did not work on our 293T's, but we had the problem of being unable to cleanly pipet the ionomycin into the 6 well plates because the wells we looked at with the scope became entirely blocked by the scope lens. Illumination of CatCh cells with 476nm laser from confocal did not increase x-rhod1 intensity. eYFP expressing cells were identified, so transfection worked. Will try again Thursday and Friday with larger plates, and possibly switch to FURA-2 and Avin's laser if the confocal still doesn't work. Anthony is going to try further with x-rhod1 dye in neurons to make sure that the dye/laser parameters aren't the problem. Will also attempt the co-culture experiment on these dates.