Question

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?

Answer 1

When subsampling, the concentration of cells in the remaining volume increases. To correct for this, calculate the final concentration by considering the dilution factor at each subsampling step.

Step-by-step explanation:

When you subsample from your rock suspension, the volume of the suspension decreases, which effectively increases the concentration of cells in the remaining volume. This means that the events/uL that the flow cytometry is showing you is not the final concentration, but rather the concentration within each subsample. To correct for this, you can calculate the final concentration by considering the dilution factor at each subsampling step. Let's take an example:

In the first subsample, you start with 0.8 mL from a 10 mL suspension. The dilution factor is 10 mL / 0.8 mL = 12.5. If the event density in the subsample is X events/uL, the final concentration can be calculated as X * 12.5 events/uL.

Repeat this calculation for each subsample, accounting for the cumulative dilution factor at each step. This will give you the corrected final concentration of cells in the suspension.