Its a problem inherent to the dye. Have been experiencing that the extent of precipitation varies from run to run which varies the concentration of neutral red in the solution. As a result, i am finding huge variations in od values from the same number of cells. Have been working on ways to minimize precipitation. As soon as i manage something, will let you know.
Have found any solution yet?
Dear Kumar, so far still no promising findings. Thank you for your kind assistance.
Do you incubate your neutral red dye solution in culture medium at 37oC prior to adding to assay plates? What i do is i prepare a 500microg/ml solution of neutral red in RPMI containing inactivated serum at 10%. Of course, my exposures are in medium containing 1% serum. I incubate the above neutral red solution at 37oC overnight. There will be a little precipitate, but not much. I spin it at 4000rpm for some 5mins, and take the clear supernatant. My cell exposure is in 200microL, when its time for neutral red assay, I add 20microL of the above neutral red soln (after centrifigution) and incubate the plate for an additional 3h. This is followed by discarding of medium, washing the cell layer a couple of times with PBS (as gently as possible) and then extracting the dye in a soln contining ethanol:h20:acetic acid in 50:49:1 ratio. This procedure has been helpful.
Dear Beng-Jin Chee,
What medium are you using? I use DMEM HIGH and I have to add Hepes at 20mM to avoid crystals formation. Did you try this?
It comes down to pH, slightly higher the neutral will cause the ammonium (-NH4+) moiety to deprotonate (-NH3) thus making the molecule non-polar i.e. not water soluble and crystallizes out of solution.
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