hello all
i stain MSC differentiated in adipocyte by oil red o , i read that i must use the isopropanol to do quantifiying , please some one can help me by sending the protocol .
thx in advance
Hello Linda,
Once you stain and photograph your cells, you can process them for Oil Red O extraction. The volume of isopropanol depends on the well type you are using but it will not matter much as you will be using same volume of isopropanol for control and differentiated cells. eg. for 12 well plate, you can pour 500ul of 100% isoparopanol (1ml per well of a 6-well plate) and incubate it for 10 minutes. Oil Red O will be extracted and now you can take out the supernatant in standard plates or eppendorff (typical ELISA format 96-well plain transparent-you can also divide your 500 volume into five wells or you can use a fresh 12-well and put 500ul supernatant in one well-take care to not take any cells) and measure absorbance at 520nm. Take care to use an isopropanol blank.
Now after OD measurement, you can plot OD values of control/diff and get the difference/significance.
Hope it helps!
Hello Linder,
the protocol for the oil red o staining of Dr. Kumar is very nice,
but we also had some difficulties by staining the differentiated cells with this method.
So we confirmed our obtained data with an immunofluoresence staining of adiponectin, visualizing nicely the lipid vacuoles of differentiated cells.
Just as a second method to quantifiy the population of differentiated cells by counting or measuring the immunofluoresence signal.
Hello andreas ,
Could explain us how we can make it in details ??
Thanks a lot
The method is quite easy, it’s an ordinary immunofluorescence staining.
The protocol in our lab:
- cells were seeded on NuncTM Lab-TekTM SlideFlask chambers and washed with PBST
- fixed with 4% paraformaldehyde containing 0.1% TritonTMX-100 for 15 min at room temperature
- washed twice with PBST for 15 minutes
- blocked with antibody dilution buffer containing 10% FCS
- stained for 1 h with a mouse monoclonal antibody against adiponectin (Abcam)
- washed twice with PBST for 15 minutes
- stained with conjugated secondary antibodies FITC-, Cy3- and Cy5 (Jackson Immunoresearch) depending on the chosen fluorescence channel
- washed twice with PBST for 15 minutes
- DAPI staining, sealing and imaging
Quantification: I. counting cells with lipid-vacuol formation vs undifferentiated cells
II. Immunofluoresence quantification by ImageJ (Treshold setting, measurement of the mean gray value and the stained area of the cell).
Hope it helps, but as I said, we just performed this assay, because we had also problems with the oil red o staining.
Ps: Sorry for the format, I wrote it in Word
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