Accessing Transfection efficiency using fluorescence microscope (Alexa Fluor 555 - (Oct/19/2012 )
I am trying to optimize how much amount of the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control (Invitrogen, Catalog number: 14750100) I should use in my experiments. I have followed the reverse transfection protocol using Lipofectamine RNAiMAX in 6-well plates. After 34 hours , I sucked out the medium and added PBS, and then went onto image each well using fluorescence microscope.
1. I used Green filter for checking the fluorescence. Is this the right filter that I should use? Please suggest.
2. How can I say the efficiency of my transfection in terms of % from fluorescence microscopic examination? Should I count the cells in a haemocytometer? Right now, I have saved images in UV mode,fluorescence mode and the merged mode. From these images, is there any way to know the transfection efficiency?
Thanks in advance
The light used for excitation of red dyes is green, so a green filter is appropriate. The easy way to remember this is that shorter wavelengths are used to excite the fluorophores which will then emit fluorescence at a longer wavelength, so reverse the usual spectrum order: ROYGBIV and you will know if you are using a filter of a shorter wavelength.
The way to assess trasnfection efficiency is to count the number of nuclei per field/image (usually stained using DAPI which emits a blue light) and count the number of (in your case red) cells showing fluoescence appropriate for your dye. From there I am sure you can work out how to do a % calculation... Make sure you include appropriate controls!!!!!