Hello.
I want to use flow cytometry for the ROS production due to the exposure of Nanoparticles. My cells are adherent cell cultures. Please suggest a detaching solution and the concentration which will not affect the ROS production. I grow cells in 6 well plate for 24 hours, then load the cells with dye, after washing the cells I add Nanopartcles to the cells and incubate them for different time points. After this I need to detach the cells for using FACS. Does the detaching solution cause the loss of Fluorescent tags (DCFDA) from the cells?
Please help
Thank you
Here we detected ROS in macrophages after the culture with MWCNTs. https://www.researchgate.net/publication/201865511_The_response_of_peritoneal_macrophages_to_dapsone_covalently_attached_on_the_surface_of_carbon_nanotubes?ev=prf_pub
Mf can be detached by a strong pipetting in NaEDTA (1-2 mM), other cells could be treated with trypsin. Even if detaching protocol have an effect on ROS production, you will have your negative (non-treated) and positive (h202) control, to see the effect of nanoparticles. Alternatively, DCFDA could be measured on fluorescence plate reader such as Synergy HT.
Best luck
Article The response of peritoneal macrophages to dapsone covalently...
For ROS study you can use either DHE or DCFDA .we have used both and got significant results in each case. Usually for attached cells we use trypsin .So, you should also check the control sample (without treatment sample).If your cells are cancer cell then there may be some ROS load but take this as control.
Briefly, treat the cells (attached) with nano particle for requisite period (24,48...etc) then detach the cells (use trypsin)
Use two controls-untreated negative and unstained (without DHE or DCFDA)
Take the cells in suspension and wash it with serum free medium once
Add DCFDA or DHE to the suspension and incubate for 15min-30min (standardize)
Wash for once and do flow
Hello
i agree with Saswat Mohapatra .
Grow 1.5 x 105 cells per experimental condition (1 well)
Collect cells in conical test tube
Stain cells with 20 µM DCFDA in 1X Buffer for 30 minutes at 37 ºC
DO NOT wash cells after staining
For Flow cytometry Measurement
1-Grow cells (adherent or non adherent) in glucose
based media so that on the day of the experiment
there are at least 1.5x104 cells per assayed condition
(treatment, dose, time). Include in the calculation
enough cells for control signal (control compound,
control vehicle and non-stained control cells). This
number takes into account any cell loss experienced
during processing.
2- Harvest cells and ensure a single cell suspension by
(A) gently pipetting up and down suspension cells or
(B) by fully detaching adherent cells (e.g. trypsinize
and quench with media).
3- Stain cells in culture media with 20 µM DCFDA and
incubate for 30 minutes at 37°C. Once the incubation
is completed, DO NOT wash the cells.
4- After staining, treat the cells with compound(s) of
interest and ensure appropriate controls are included.
If Tert-butyl hydroperoxide is chosen as the positive
control compound, optimal signal is obtained after 4
hours of treatment.
5- Gently pipette cells up/down to ensure single cell
suspension.
6- Analyze on flow cytometer. Establish forward and side
scatter gates to exclude debris and cellular aggregates
from analysis.
7- DCF should be excited by the 488 nm laser and
should be detected at 535 nm (typically FL1).
8- Ideally 10,000 cells should be analyzed per
experimental condition. Cells should not be overly
dense during the experiment (< 1x106 cells/mL).
To ensure the best results , suggested the following :
1- Treat cells for desired period of time
2-Read signal at Ex485 nm/Em535 nm Determine change as percentage of control after background subtraction
Good Luck
thank you all
Can i know little more about the DHE staining? I am not familiar with that. can you suggest me some papers which have used DHE staining. Thank you so much
I read about DHE, does this stain only show us the oxidative stress in nucleus or its also for cytoplasm? and can DHE staining be used for Microplate reader as well?
and what should be the cell count per well for plate reader experiments?
i use 10,000 cells per cm Sq area
You could try using UpCell plates so that you do not need to digest the cells.
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