julnobody

Hi,


I was wondering about Nile Red storage after reconstitution in DMSO, most people aliquot it and keep it a -20C but I have also seen people storing it at room temperature in the dark.

Are both of these solutions ok ?

Thank you

Hi,


I am doing Nile red staining on glioma cells,
- harvesting around 80000cells with 0,5% trypsin EDTA,
- inactivation with FBS, transfer to FACS tubes
- spinning 350g for 5mns,
- washing with 2mL PBS,
- spinning 350g for 5mns,
- adding 500uL Nile red working solution for 15mn
- spinning 350g for 5mns,
- washing in PBS 1x,
- respinning and finally resuspending in 200uL PBs for FACS analysis.

It is a lot of centrifugation steps which probably accounts for the fact that I have very few cells during FACS analysis, has anyone any other protocol that could enable recovering more cells ? (I'd like to add that I carefully remove the sup after each centrifugation step)


Many thanks

Hi,

Very simple stuff but I am annoyed with it,
I am trying to stain lipid droplets in cells using Oil Red O.
I had a previous batch of the stock solution that was perfect, no aggregates, very clean, and nice staining but I have tried to prepare some new from powder (0,5g in 100mL isopropanol), heating overnight at 56C and filtering twice in whatman paper but even with this and after additional filtration, I get a lot of aggreagates on my cells when observing in microscope.

I prepare my working solution from stock (I tried 6:4 and 3:1 ORO/water ratio) used immediately with and without filtering before adding to the slides, I wash with water several times but it doesnt seem to help

The solution I make, 0,5g ORO in 100mL isopropanol, seems very dark compared to the previous one but I think this is the usual concentration though....

If anyone has a way of doing it that works, I'd be pleased to know about

Thanks

Hi,

I am planning on doing lipid analysis on cells, tissue (fresh frozen) and parrafin embedded.

Among other things I want to perform a cholesterol level measurement, is it possible to do so by lipid extraction from formalin-fixed parrafin-embedded tissue ? (Fresh frozen is probably the best but I have more material parrafin embedded)

Thank you;

Hi,

I would need to do section (preferably longitudinal) of U87 cells for electron microscopy, I was wondering how thick (approximatively)  U87 are when adhered on a plate ?

Because I can't find the info anywhere and I would need the info quite quickly so I have no time to do z-stacks of in in confocal.

If anyone has an idea please help!

thanks