ROS assay in RAW 264.7 Cells.

More Lokesh Sharma's questions See All

Is eritoran available for research use in US?

I am looking to use eritoran for blocking TLR4 in my experimental model, but I could not find it available from any commercial company. Can you suggest where I can purchase or how easy it is to...

04 May 2014 5,284 3 View

Which of the Covid-19 Vaccines would elicit the best immunoprotection and why?

I want to know which of the available Covid-19 Vaccines would elicit the best immunological response and why

02 March 2021 1,388 3 View

No title

02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Which fluorescent dye to use for measuring total ROS production?

Is DCFDA appropriate for measuring total ROS production, or can it only detect certain types of ROS? If I want to measure total ROS production, which dye should I use?

01 March 2021 9,447 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

No title

28 February 2021 9,936 2 View

Adam P. Gunn

Hi Lokesh,

In my experience the DCF assay gave the best signal to noise when the cells were washed and the fluorescence readings taken in HBSS or PBS. The vitamins in DMEM and other complete media can be autofluorescent and also quench some fluorescence signals, causing poor signal/noise. I also only needed to incubate the cells with the oxidants for 2 - 4 hours to see a good signal (also not good to keep in HBSS too long as they can die, but you can keep in normal media and exchange to HBSS before the read). Are you using a positive control (e.g. H2O2, Sin1, Cu+ascorbate)?

Fabianno Ferreira Dutra

hi lokesh,

do you really need to stimulate for 24hs? if so, i recommend you to load the dye at the end of the experiment. i would start doing a kinetic experiment with your stimulator. some stimuli induces ros generation as fast as 10min. use pma 100nM for 10-30min as a positive control. it always works.

if you are using facs: you can make stimulations with serum and any cell medium such as rpmi and dmem but it is important to load the cells with the dye in the absence of serum. i use rpmi and it works fine. after loading, wash the cells and ressuspend them in pbs.

if you are working with a fluorometer: preload the cells with the dye and work with mediums without phenol red.

good luck!!!

Lokesh Sharma

Thanks Adam and Fabiannno for your answers. I think both of you are pointing at important drawback in my protocol. I have been using media containing 10% FBS in the entire assay even while lysing the cells I used to add 2x lysis buffer in serum containing media. I will work on your suggestions and will let you know soon about the results. Thanks a lot again to both of you.