Hi friends,
I am trying to determine ROS by FACS analysis.
The protocol which I followed here,
In this experiment I used colon cancer cell (HCT116)
I used as a positive control H2O2 treated cells (100uM, 1 h treatment)
1. After treatment the cell were washed with PBS and incubated with 30uM 2’,7’ –dichlorofluorescin diacetate (DCFDA-Sigma) for 30 mins.
2. After incubation the cell were washed with PBS and trypsinised and collected in PBS and keep in ice until FACS analysis.
After staining with DCFDA, When I try to acquire the data by FACS, I am getting strange data.
Usually, H2O2 treated cell should shift to right side, for me in contrast it shifting to left side. I dont know why its happening for me?
I have attached image with all condition of FACS setting.
Please find the attachment.
Hope to get some positive reply from every one.
why it shifting to left side?
How was your gating strategy? Did you eliminate doublets? Did you use a viability dye?
Thanks for your reply Andre Fernandes,
There was single population, cells were healthy.
Hi Pasupathi
You should increase your H2O2 exposure times. Try a kinetic of H2O2 (2h, 4h and 6h). You can and should also titrate H2O2 concentration (100, 200 and 400uM).
As for the flow cytometry analysis you should
- eliminate doublets by a FSC-H vs FSC-A (you need a single cell analysis and during flow acquisition more than one cell can pass through the laser).
- gate on live viable cells using PI or 7AAD negative population (H2O2 can induce apoptosis and dead cells have autofluorescence)
Try to detect ROS using Hitachi spectrofluorimeter
(excitation 480 nm, emission 520 nm).
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