Talk:Cell count and normalization in HRR
Feedback from Sabine (email 2020-09-23)
General I think it should be pointed out that Nce refers to the total cell count, i.e. the sum of viable cells (Nvce) and dead cells (Ndce). I know this is explained here https://wiki.oroboros.at/index.php/Living_cells#The_units_of_the_cell_count_and_cell-count_concentration, so maybe this link should be implemented. In my experiments I always normalized to the number of viable cells, which I think is also an option. No matter whether the Nce or Nvce is used, the viability should be >95% and comparable between samples. Therefore I would suggest to include the viability in the excel sheet. Not for calculation but for documentation.
→ The link should be implemented → The question is whether "dead cells" are cells with functional or dysfunctional mitochondria (when and how did the cell die. In media or in MiR05?)
Complete replacement “The cell-count concentration Cce calculated from the initially determined Cce,σ (3) can be compared with the median of the finally determined cell-count concentrations Cce.i (13) for quality control of cell counting.” In my understanding, this is not (only) a quality control of cell counting but a quality control of proper dilution.
Pipetting into V#-VJ.i Why are no subsamples taken from the chamber? (Cell count concentration control and further assays) I think a control of the cell count concentration in the experimental chamber would be important due to the higher dilution factor (compared to the complete replacement) and the removal of medium with the volume Vj,I which bears another risk of error due to the additional pipetting step. Pipetting into V+VStopper capillary Same as before: Why are no subsamples taken from the chamber? (Cell count concentration control and further assays) excess volume: VStopper capillary + sampling + waste total volume before subsampling from chamber and closing chamber This implies that subsamples are taken from the chamber but it is not mentioned in the text or shown in the figure. I think it could be easily implemented as the total volume V# is 2.59 mL before closing the chamber.
→ The scenarios shown in Figures 2 to 4 are just examples for different possibilities and not fixed protocols. For sure, also for methods 2 and 3, subsamples can be implemented. The number and volume of subsamples is dependent on the experimental design, e.g. further assays, and the amount of available sample. The amount of human blood cells is restriced and might not allow for taking subsamples.
Feedback from Carolina (email 2020-09-25)
2. Cce = 1 Mx/mL. By reading the text and looking the Excel file, I did not understood from where the value of Cce = 1 Mx/mL comes. I would like to ask you to be sure. Is it an example of measured cell concentration (Cce) of a sample from the O2k chamber? When I arrived to the calculation for Cce,J I used the value for Cce, σ and then I related that I was wrong and the value used here should be the one corresponding to Cce. I was confused because following the figure (for example Fig. 1) the step for taking the sample from the respirometric chamber for cell-count is the step 12, and the calculation of Cce,J comes before
→ Cce is not a variable in the calculations, but a parameter that has to be decided based on respirometric background information.