To screen some of my compounds for its anti-inflammatory property; I primarily tested those in RAW264.7cell lines by inducing LPS(10ug/ml) to release ROS & NO (by fluorescent method and Griess reagent method respectively) . Most of the times I observed any significant NO & ROS released from RAW cells to compare in normal cells and LPS-induced cells. To find a solution, I tried with different concentration of LPS (with different manufacture batch) in different passage of RAW cells. I experienced the same again and again.
1. Can you please suggest any other cell lines for ROS & NO release experiment (I tried in V79 cell lines too)?
2. Can you please suggest some alternate method to test for ROS & NO estimation in RAW cells?
Hi Chandra! I can suggest to detect the NO release by chemiluminescence. You can use a chemiluminometer, both for an on line detection of NO, or for the NO2- detection. This tecnique is more sensitive with respect the Griess method (LLOQ 0.5 pmol), and on the basis of my experience, more appreciate by the referees. You can find the details on the attaches.
Best regards,
Fabrizio
Hello Chandra
regarding ROS measurement, an old but reliable technique I have used extensively is the reduction of NBT (nitroblue tetrazolium) into the coloured compond formazan which can be measured spectrophotometrically. For NO, I also use the Griess method.
Two references from our group which you will be able to find on line are
(1) Gómez, L.A., Cortés, R. & Smith, C.T. 2013. Effect of PHA and LPS on the Activity of Nonspecific Cytotoxic Cells and Macrophages of Rainbow Trout (Oncorhynchus mykiss). Gayana 77(1): 53-59.
(2) Gómez, L.A., Cortés, R., Valenzuela, A. & Smith, C.T. 2013. Kinetics of reactive oxygen species produced by rainbow trout (Oncorhynchus mykiss) leukocytes and the effect of the antioxidant astaxanthin. Acta Vet. Brno 82: 435-439.
If you have any further questions, please fell free to ask me
Carlos T. Smith
If you are working with cell line alone there are several probes available for ROS estimation DCFH-DA (probably you would have used this) is the best option if you are still not getting results that might happen because of leaching out of fluorophore you may order a modified version of the dye called CM-DCFH-DA it works much better and use with both flurimetric or flow cytometric methods. Further, for nitric oxide and its derivatives a fluorescenct probe named DAF-FM is available with life technologies. It also works find and relative quantification may be done with microscopy or flow cytometry comprehensively.
Best
Hi Chandra.
You might want to try RAW264.7 cells, TIB-71. I've tried and successfully induced the cells with only 1ug/mL of LPS. Some RAW264.7 cell lines might require both interferon gamma and LPS for a successful induction.
I've attached here a link to a publication that you might find it useful.
Good luck!
http://www.hindawi.com/journals/ecam/2013/304713/
Hi Chandra,
after how many hours from LPS do you detected ROS and NO release? Generally in J774A.1 macrophage, cells similar to RAW264.7, I detect a significant NO and ROS release after 22-24 hours.
Best
i would suggest to check the passage number of your cell line while culturing cells for nitric oxide detection by griess assay. Beyond 10 passages the amount of NO gets decreased significantly.Since the amount of nitric oxide depends on cell number and conc of LPS, you can try with different cell population per well like 50,000-2 lakhs with 1-5ug/mL of LPS.
Dear Dr. Carlos Smith, Dr. Aditya, Dr. Pui-Mun Chan, Dr Stefania Marzocco, Dr Madhuvanthi Chandhrakanthan...
Thank you very much for your valuable suggestions, the alternated methods and cell lines along with the related references.
Regards
Did you checked, if the LPS you used really activates your RAW macrophages? I would check the TLR-binding of your LPS.
For ROS measurement DCFDA is good enough, to get good results.
Probably increase the LPS concentration and set up your experiments for 24h - 48h to see an effect. For NO measurement Griess is a good method. But if you have the opportunity, use a Clark-type NO electrode
Hi Chandrashekhar and Carlos,
Since I am going to start such screening assay for a set of compounds, I want to ask you a few questions such as:
which culture plate do you use for your assays - 96, 48, or 24 well plate?
What cell number do you take in each plate?
How long do you keep the cells stimulated with LPS?
Did you try ELISA for cytokines? If yes, can you please share the protocol with me, or the key points as
Dear Deabsis,
Preferable - 96-well plate (as per your convenience and lab facility).
Cell number - 50,000 cells per/100uL/well (for 96-well plate)
LPS treatment - min of 20h and max 22h (standardise at your lab).
Wish you good luck.
One of my experiences tells that 100ul of media leads to starvation of cells if incubated for around 20 hours... Didn't you observe this?
Dear Debasis,
Do you mean only media (without FBS)? If so, yes. But in
No, I used 10% FBS only, however my cell density was also high... got it..
10 ug/mL LPS is almost a supraphysiological dose. 1 ug/mL would be more acceptable in literature as a max dose. In species like Salmonella t., 5 ng/mL LPS is already quite potent.
I used 200 - 500 ng/mL Sal. typhimurium LPS for ROS/RNS induction.
If you want to detect NO, try DAF-FM, an established probe sold by Invitrogen. It takes a wash step and about extra 15-20 min wait for oxidation of the fluorescent product before readout.
RAW264.7 cells are already quite robust and easy to use. The best time frame to capture NO is 8 h to 18 h (reflecting iNOS expression).
As for ROS (superoxide), a similar time frame is recommended. Beyond 18h through 24h, the ROS/RNS levels could actually wane as cell death and negative feedback set in. Good luck.