I am a newbie in FACS analysis: so I used this reagent from dojindo called cell cycle blue reagent and do staining in live cells (not like PI) they stated that fixation is not needeed, the problem is this reagent has not been used in available paper for my cell lines (min6 insulinoma cell lines), thus I don't know why my result is like this... I tried to analyse in FlowJo but I couldn't get the result of the cell cycle that is usually shown. 1st peak G0/G1, then s phase, then G2/M phase... what went wrong?
I attached some of my result:
I went through your PDF data .
1. Plot 1 -SSC VS FSC- reduce the input voltage of FSC and SSC and try to bring the population within the axis. Though, it looks that it has two population, after adjusting the threshold and FSC, SSC voltage, it may show one population and gate that population.
2. Doublet discrimination plot - pacific blue A Vs pacific blue H - first try to reduce the input voltage of pacific blue during data acquiring and it will bring your green and orange population in one place/ overlapping condition and then again gate the singlet population and plot count Vs pacific blue A.
3. Also , reduce the cell number to do the cell cycle analysis.
I took your FCS data gated "all events" to include both the populations that appear on your FSC vs SSC plot. It is possible that these cells appear differently on this plot in G2 vs. G1 because they could be bigger, more granular, whatever. If you do this and constrain the G1 gate you can get this cell cycle model. Really you need to include some control compounds/ treatments that arrest the cells at different parts of the cell cycle to know if this is legit or not though.
Based on the screenshot you showed, I think you need to gate out some debris in the sample, and constrain the G1 and/or G2 with a gate. But again, without seeing some controls its hard to know what is real.
https://docs.flowjo.com/flowjo/experiment-based-platforms/cell-cycle-univariate/
I set the gate in the image "Gate 1"
Opened the population, set the axis for Pacific Blue
Open "Cell cycle" set gates for G1 and G2. When you set them it should come up with the option for "Constrain G1" or "Constrain G2". Then the calculations worked. However, it is hard to know if those numbers accurately reflect the cell cycle stages of the cells without controls.