Details for amaxa FAQ article #50

«How do you determine cell death?»

We determine cell death in two ways:


1) FACS determination of viable/dead cells by PROPIDIUM IODIDE STAINING.
We normally analyze transfection efficiency in living cells by FACS: We first exclude cellular debris by gating for the "normal" population (regarding size and granularity) in the forward-sidescatter. From this gated population we determine dying cells by propidium iodide staining and exclude them from analysis by setting another gate. So, only those cells which are in the FSC-SSC gate and are not propidium iodide positive are analyzed for efficiency.
To be even more precise in the determination of the mortality for adherent cells, we also collect the detached cells in the supernatant and combine these after trypsinization with the former adherent cells and include them in the FACS analysis. This helps ensure that our data is complete and accurate.

2) FACS determination of total cell loss induced by Nucleofection (optional method):
In order to get an idea about the total cell loss, we compare the initial cell number per sample with the final cell number per sample.
For that we use APC-labelled beads from BD Biosciences (to detect APC your FACS needs a 4 channel laser, because APC is detected on channel 4).
We prepare a stock of beads (1000 beads/µl in PBS). After 5 minutes of vortexing, we add 50 µl of the beads to the sample we want to analyze by FACS.
To count the beads by FACS you have to lower the threshold in the FSC/SSC down to 20 because they are much smaller than cells.
After analysis, we do the following calculation to define the number of used living cells in the analyzed sample (input X, unknown value):

input X = number of used beads*/counted beads x counted cells in R2**

* input: 50µl = 50000 beads
**R2 gate means: normal cell population in FSC/SSC without cellular debris (gate R1) AND without PI-postive cells in FL-2/FL-3

By comparing the X-values for the untreated sample and the nucleofected samples you can calculate how many dead cells you have in your treated samples.