# Concentrating cell concentration after subsampling

I need some help with regards to a concentration correction. I am measuring how many cells detach from a rock vs sonication time. Becuase I start from a rock, I cannot make aliquots and measure independent samples. So I put my rock in 10 mL of PBS and and sonicat for 1 min and sampled 0.8 mL. With this sample I measure cell density using flow cytometry. I then placed the rock to sonicate for another 2 min (a cumulative time of 3 min of sonication) and subsampled 0.8 mLs again. I did this for a cumulative time of 30 min, with subsampling at 1, 3, 5, 10, 15 and 30 min. This means that I was left with 5.2 mL of sample after my final subsampling (0.8 mL per subsample * 6 = 4.8 mL; 10 mL - 4.8 mL = 5.2 mL).

I measured my cell density using Flow cytometry and was given a events/uL concentration. But I think that every time I am subsampling I am effectively increasing the concentration (events/uL) because my cells are being resuspended from the rock into an ever decreasing suspension. Is this correct or will the events/uL that the FCM is showing me the final concentration independent of this diminishing volume? If not, how do I correct for this to get a comparable events/uL?

Here is an example of the conctrations over time after a cumulative 10 min of sonication (i could not figure out how to insert a table, hence in picture format)

So the solution for this can be in two ways:

1. calculate the total number of events (evenst/uL * total volume of sample at time X) and then devide by the initial volume.

2. or perform a correction factor, by calculating the volume reduction factor (subsample volume / volume before subsampling) e.g. at 60 seconds: 0.08/10. You then calculate the reciprocal of this. and multiply by events/uL

NB: In these calculations is not included the fact that cells are being removed in each step, and as such there is still some dilution happening.