Hi Everyone,
I have problem with Neutral Red solubility in FBS free medium. I made 50uG/mL STOCK and incubated 24h, what (always) results in small crystals. I tried to centrifuge and filter BUT after filtration signal decreased (especially after 0.45uM nylon filter).
Any ideas? :)
THX for suggestions!
Have you tried heating the solution slightly?
If it were me...I'd heat it to about 40 Celcius while stirring. After it dissolves, it should stay dissolved.
It's like making sweet tea here in the 'Deep South.' We put too much sugar, but the only way to get 'too much' sugar into solution is to add the sugar while the water is hot... If you add the sugar while the water is still cold, it doesn't dissolve completely.
I forgot to add, that I incubate it 24h in 37C and this process makes crystals.
Today I've done something different - made a STOCK, incubated only 1.5h, filtered through 0,22uM fillter, added to cells and after 3h I'll read the plate - I'll see if the signal increases or not :)
I observed the cells under microscope and everything is fine - no crystals and more NR in cells.
BTW: Thx for answear!
We'll see :) BTW: Yesterday results were good - so I don't know what for should I incubate it 24h in 37C (each protocol says it).
Hi Kamil,
I'm having the same problem over here. Are you using a spectophotometer to read the absorbance? If so, could you please tell me (in case you have the data) the absolute optical density you observed in a confluente monolayer of cells in a 96 well plate?
I am aiming to get OD> 0.4 since Im following an OECD guideline and that is an acceptance criteria.
Thanks.
Hi Violeta,
Sorry for late answer your qestion. I found only data from my first NRU experiment for HepG2 that I've done. Max OD (540nm blank corrected) I've obtained was 0,663. The other values varied from 0,587 to 0,663.
I hope it will be useful for you :)
I incubate my NR solution, overnight at 37, centrifuge it after (1800 rpm, 10min) and there are no crystals visible.
Do you wash your plates after incubation with NR, or just aspirate the medium?
If u wash how many times?
I noticed that the wash is decreasing my absorbance intensity (around or less than 0.1) and only with much larger cell number (>500000) i get signal around 0.3. However without wash I have the same trend of cell viability as when I wash them, just with much better absorbance..
any suggestions?
p.s. I use primary cells (isolated macrophages/lymphocytes)
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