Colony formation in soft Agar assay

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eliassii
eliassii's picture
Colony formation in soft Agar assay

Hi,
I'm using LMP Agarose (AgarPlaque Plus Agarose, BD Bioscience) for the assay.
Lower layer: 0.5%
Upper layer: 0.4%

I'm trying to overcome few problems with the soft Agar assay:
1) Althoug I place the plate at 4C for 20min, most of the cells concentrate on top of the lower layer.
How could I overcome the cell spreading problem?
Maybe by adding reagent that would give higher Viscosity to the upper layer and by that maintain the cell scattered evenly in the upper matrix until Gelation??
2) After colonies appearance I want to use MTT Colorimetric method to quantify the live cells. How could I dissolve the solid matrix w.o. harming the mitochondrial Enzymatical activity?

I hope that simple solutions are avilable to solve those problems?

Thanks in advance,

Ilan

marcus muench
marcus muench's picture
What volumes and plate sizes

What volumes and plate sizes are you using? Generally it is desirable to have the cells in a narrow plane to make counting easier (by avoiding the need to continuously refocus on different focal planes). I place cells in 0.5ml upper layer over a 1ml underlayer in 35mm2 plates and, for 60mm2 plates I use 1ml on top and 2 ml at the bottom.

If you plate on cold solidified underlayers, you could get the upperlayer to solidify faster.

I don't think agarose will allow you to harvest the cells with any ease. If your cells grow in methyl-cellulose, that can be scooped up and diluted to spin out the cells.

eliassii
eliassii's picture
Hi Carson,

Hi Carson,
I'm using 96w TC plate format:
Lower layer: 0.5% 75ul
Upper layer: 0.4% 50ul

I'm trying to overcome few problems with the soft Agar assay:
1) Althoug I place the plate at 4C for 20min, most of the cells concentrate on top of the lower layer.
How could I overcome the cell spreading problem?

2) After colonies appearance I want to use MTT Colorimetric method to quantify the total live cells in different treatments. How could I dissolve the solid matrix w.o. harming the mitochondrial Enzymatical activity?

I hope that simple solutions are avilable to solve those problems?

Thanks in advance,

Ilan

marcus muench
marcus muench's picture
1) As I tried to suggest in

1) As I tried to suggest in my last post, most of the time in my experience with hematopoietic progenitors it is desirable to have the cells concentrated, although I've never had them settle out completely. I've never worked with such small wells, it is possible that the static charges and meniscus effect of the wells is causing your cells to accumulate in a certain spot. Generally in larger plates the problem is that the matrix is thicker at the edges, which causes cells to be distributed at different levels compared to the center of the well.

If you want the cells more spread out, can you just plate them in a larger volume in a single layer or, at least, shrink the size of the lower layer and expand the upper?

2) Again, I have never stained with MTT in agarose, only liquid cultures. I'm not sure of a good method to accomplish this aim.

eliassii
eliassii's picture
Hi Carson,

Hi Carson,

first of all thanks for the advise.
I'll try to seed the target cells in pre-chilled plate in order to accelerate the Gelation (lower cell sedementation).
As for the Colorimetric method to quantify the total live cells in different treatments I found more friendly substrate WST1. And as we "speak" preliminary results indicates that there is no need to dissolve the solid matrix in order to reach the mitochondrial Enzymatical activity?

Thanks in advance and good luck,

Ilan

marcus muench
marcus muench's picture
Thanks for letting us know

Thanks for letting us know what is working for you. It is nice to hear feedback so we all can learn something.

Orest
Orest's picture
Maybe it's too late already,

Maybe it's too late already, but I have tried FDA/EtBr and AO/EtBr stainings to visualize live cells in soft agar under Fluorescent microscope (0.4% agarose, carcinoma cell line):
- Ethidium bromide staining will only enter dead cells and it fluoresces red under the FL microscope;
- Acridin orange (AO) will enter all cells (but EtBr still predominates) and stains cells FL-green;
- Fluorescein diacetate will be enzymatically converted to green FL stain but count cells quickly because it fades very fast.

For procedures, please refer to Liviac 2011
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TGF-513F91K-3&_user=464575&_coverDate=01%2F30%2F2011&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000022258&_version=1&_urlVersion=0&_userid=464575&md5=bfe1af27548c796edf7f6e35ac82824c&searchtype=a

 Mather and Roberts,
http://books.google.at/books?id=Cla-N2x4cRQC&pg=PA74&dq=acridin+orange+ethidium+bromide+viability&hl=de&ei=4fGBTdWwB47Nswaw5PGmAw&sa=X&oi=book_result&ct=result&resnum=1&ved=0CCoQ6AEwAA#v=onepage&q&f=false

protocol-online.org
http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3841

suchitraananth
suchitraananth's picture
Hi Ilan,

Hi Ilan,
Try to use Alamar Blue from Invitrogen.  It is non-toxic and amenable to plate readers such as the Tecan.  After adding 10% of the total volume (so 12.5 uL Alamar Blue), you can incubate the plates in 37C for about 1-4 hours for most cell lines and densities.  You can measure viable cells with the fluorescence plate reader or colorimetric plate reader.  You can test different incubation times, i.e. 1, 2, 4, 6, 24 hrs for each cell line and density.  Try to include blank samples that only have media.  If you have a positive and negative control in your plate, it should be easy to normalize the samples to the non-treated or non-targeting control after subtracting background fluorescence from media only.