OK, so I think I have worked out the problem. Its the fixation step. After fixing with 3% PFA for 20 min at room temp, the matrigel had depolymerised and formed back into a semi-viscous state. A bit of searching on the web led me to the BD Biosciences FAQ for their matrigel (which I am using).....http://www.bdbiosciences.com/documents/BD_CellCulture_Matrigel_FAQ.pdf

It says sometimes the addition of glutaraldehyde is needed to prevent this from occuring. But this can add massive background for IF microscopy so need to quench with NaBH4 to prevent this. I will have a go and see if this helps and I can still see my antibody staining:

"How can I fix cells in BD Matrigel matrix for applications requiring sectioning for subsequent immunohistochemical or immunofluorescence experiments? How can I avoid BD Matrigel matrix de-polymerization after fixation?

Fix BD Matrigel matrix with 2% paraformaldehyde. In some cases BD Matrigel matrix tends to de-polymerize after fixation. Adding 1% glutaraldehyde to the BD Matrigel matrix can prevent de-polymerization.

Glutaraldehyde is a fixative for electron microscopy and tends to generate significant background auto-fluorescence. We recommend adding a quenching step utilizing NaBH4 after fixation (for immunofluorescence assays). Since NaBH4 generates air bubbles in the process, this step should be performed on the bench without any shaking to minimize bubble formation. You can also try 0.1% to 0.5% glutaraldehyde to minimize or prevent depolymerization. The use of less glutaraldehyde may produce less background fluorescence."

Have a look to the concentration of your matrigel, it's maybe too low and your cells are not well attached to it...(or maybe your matrigel is disturbed by all these treatments..??)

I would check at each step as you said the number of cells remaining on the slide to have an idea about the problem...

Best of luck.

Did you see if the loss it's increassed after tween? if the detergent is solubilizing the matrigel... i would try to fix and permeabilize at the same time only using with methanol ...

good luck!

Thanks for your suggestions. Laura I think it may be the tween step (or indeed the T-X100 permeablisation). I have tried methanol fixation and that was also not good for the matrigel and solubilised most of the sample away. I havent tried 1:1 acetone:methanol fix yet, but fear the majority of my antibodies I am staining with will not work with this fixation - I will look more closely next time to see after each step how many cells are left

Senthil Muthiswamy's lab website had detailed protocols for 3D culture and staining. That might be useful for your studies http://muthuswamylab.cshl.edu/ml_protocols.html

Thanks ashok the website is pretty much the same as the Brugge lab protocol website and debnath et al methods paper describing the 3-D assay that I am already following. I suddenly thought also I am storing my fixed slides at 4oC which I do with all my IF. This may also e degrading the matrigel!

True Chris !!! Do not store your slides at 4°C it degrades the matrigel !! I would suggest instead to store them at -20°C but I must admit that I don't know if it will have an impact on your cells...

OK, so I think I have worked out the problem. Its the fixation step. After fixing with 3% PFA for 20 min at room temp, the matrigel had depolymerised and formed back into a semi-viscous state. A bit of searching on the web led me to the BD Biosciences FAQ for their matrigel (which I am using).....http://www.bdbiosciences.com/documents/BD_CellCulture_Matrigel_FAQ.pdf

It says sometimes the addition of glutaraldehyde is needed to prevent this from occuring. But this can add massive background for IF microscopy so need to quench with NaBH4 to prevent this. I will have a go and see if this helps and I can still see my antibody staining:

"How can I fix cells in BD Matrigel matrix for applications requiring sectioning for subsequent immunohistochemical or immunofluorescence experiments? How can I avoid BD Matrigel matrix de-polymerization after fixation?

Fix BD Matrigel matrix with 2% paraformaldehyde. In some cases BD Matrigel matrix tends to de-polymerize after fixation. Adding 1% glutaraldehyde to the BD Matrigel matrix can prevent de-polymerization.

Glutaraldehyde is a fixative for electron microscopy and tends to generate significant background auto-fluorescence. We recommend adding a quenching step utilizing NaBH4 after fixation (for immunofluorescence assays). Since NaBH4 generates air bubbles in the process, this step should be performed on the bench without any shaking to minimize bubble formation. You can also try 0.1% to 0.5% glutaraldehyde to minimize or prevent depolymerization. The use of less glutaraldehyde may produce less background fluorescence."

When I used Matirgel (1999), the company also sold a special buffered solution for removing the cells from Matrigel-it was called Matrisperse. It worked well, and I got good cell recovery. The cells kept clumping-so I could not get accurate viable count. Then, I just decided to replate them on plastic to see if they would attach and grow....that proved that they survived the Matrisperse treatment .

Hi Chris.. I am trying to culture mammospheres ( MCF-7 line) on matrigel.. but I am not sure if I am successful.. my first question is "are they going to form suspension acinis? or the acinis will attach to matrigel?" second one ; " if it will be a suspension culture and if some are still adherent how am I going to change the media? "

Thank you

I must admit I have only used the MCF10A, MCF10A/T and the MCF10CA1 cell lines. However I believe the MCF7's should be similar. The acinis should attach to the matrigel as the cells sink to the bottom of the well (where the solid matrigel layer is) and are also surrounded by the matrigel-containing media (hence the 3-D nature of culturing).

They are fairly adherent, and by changing the media you should not dislodge too many of them. The issue is (as you may see) that when you fix the cells, the matrigel is dissolved and this is the step you will lose a lot of sample. But if you do not require this (i.e. are imaging the cells live) then this will not be a problem.

In any case (and I am not working for these guys!) the company Ibidi sell lots of different chamber slides (http://ibidi.com/xtproducts/en/ibidi-Labware/Open-Slides-Dishes:-ibidi-Standard-Bottom/m-Slide-8-well) for imaging mammospheres on both upright and inverted microscopes. They use microscope grade plastics instead of glass meaning you can image at high resolutions on an inverted scope.

Another thought is you can also grown your cells completely embedded in matrigel (rather than the overlay assay you are using currently). Then your cells will definitely not be washed away when are changing media.

Good luck!

hi Chris;

Thank you for your answer but I still have questions for you.

First of all as I understand from your answer you do not have suspension culture then.. your cells grow to acinis on the matrigel. They don NOT float.. DO I understand right? Also I am trying to have Cancer Stem Cells. Do you recon matrigel affects differentiation..?

Sorry to interrupt you so much

Thank you

Thank you so much for asking this question, Chris, and for answering everyone! I just did a side by side comparison of 4% PFA versus 4% PFA + 1% gluteraldehyde, and the difference is amazing: globs of matrigel and MDCK cysts are floating about in the PFA fix but everything stayed put with gluteraldehyde. So weird! I hope the background fluorescence isn't too awful, but will try the sodium borohydrate

Chris and Sara, thanks a lot for posting your question/comments. I'm just about to start staining spheroids cultured in matrigel. I will definitely be using the Glutaraldehyde. My only concern is that I'm doing IF staining, did you get a lot of background with the GA?

To bring an old thread back to life...Alejandro and Sara, did you notice much background noise when using 1% glutaraldehyde?