Soft Agar Colony Formation Assay

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Seagull
Seagull's picture
Soft Agar Colony Formation Assay

Hello. Does anyone know that why there are many debris around the cell colonies in the soft agar? I have not seen this phenomenon before. Thanks a lot.

samm
samm's picture
Are they some kind of

Are they some kind of satellite colonies - can they be stained/regrown?

Jason King
Jason King's picture
Seagull wrote:Hello. Does

Seagull wrote:

Hello. Does anyone know that why there are many debris around the cell colonies in the soft agar? I have not seen this phenomenon before. Thanks a lot.

Are your cells under selection or have they been infected with (Ad) virus? Or was the agar a little too hot as it was overlayered.

Seagull
Seagull's picture
To Samm,

To Samm,
First of all, thank you very much for replying.
They are sand like spots surrounding the colony. I doubt if the cells go to apoptosis?
Yes, they can be stained.

Seagull
Seagull's picture
to Parvoman,

to Parvoman,
The cell were transfected with siRNA the day before setting the soft agar assay. I use 43 degree waterbath for keeping the medium and ager. Is that OK?
Thanks a lot.

marcus muench
marcus muench's picture
Can it be contamination

Can it be contamination originating from your cells? It is either that or your cells grew and then died. What cells are these?

BTW, I've spent years growing hematopoietic colonies and we gave up hot agar long ago. Instead we use low melting point agarose. Once diluted in warm medium to its final concentration (0.36-0.5%) it will not gel for quite some time if the lab is not too cold.

Seagull
Seagull's picture
It seems more possible the

It seems more possible the second reason because, not all the colonies look like this way, and the longer the colonies grow the more colonies become this way.

Thank you for the suggestion of low melting point agarose, I can try it later. But, the problem is the agar works well for me before. This situation just happend recently. May it because I'v changed the brand of agar, which for some reason seems more solid than previous one at the same concentration (0.4%) ?

marcus muench
marcus muench's picture
As I think about what your

As I think about what your describing, I may have seen this as well with hematopoietic colonies. I attributed it to dying cells, but you see all kinds of odd little things after fewing thousands of colonies.

If your agar preparation differs, that may be it. Differences in purity etc of different preparations can very likely affect your cell growth.

If your interested, colonies can be stained forlive cells by exposure for about 12 hours to 0.5ml of 1mg/ml p-iodonitrotetrazolium violet (Sigma Chemical Co.) in water. You can fix with 0.5ml of 3% acetic acid. Live cells turn dark color. If you can grow cells under the old and new conditions, then you can see if the cells are dying prematurely in the new medium/agar.

BTW, we use SeaPlaque agarose from FMC, Rockland, ME.

Jason King
Jason King's picture
I use the same (FMC LMP)

I use the same (FMC LMP) agarose for overlayering 293 cells that have just been adenovirus infected. I don't think the 43 degree temperature would have been to blame.

Did the surrounding grains look like cell debris or were they smaller? If they were smaller, it could have something to do with the siRNA transfection reagent. Did you do transfection controls using the same reagent with a control siRNA or in the absence or siRNA? I've recently tested out about 10 different reagents, all have slightly different mixing and incubation times. I found that (like standard calcium phosphate transfection of cells with plasmid DNA), if you leave the siRNA+transfection reagent(s) for too long prior to adding it to the cells, then it can form visible complexes. Usually these are visible the next day, not immediately after adding the transfection mix to the cells.

If the grains were big enough to be debris, then I guess the questions would be: Are these cells particularly sensitive to siRNA transfection? If not, have they just become more sensitive as a result of a low level mycoplasm infection? And finally, is the gene you're targeting with the siRNA essential for survival?

Good luck.

Seagull
Seagull's picture
The analysis and experience

The analysis and experience from both of you help me a lot. Thanks!

The surrounding grains are the same size, which I think are bigger than the siRNA+transfection reagent complexes that I've noticed in my transfection, too. It happened in all the samples, including non-transfected control, reagent control and siRNA control. The breast cancer cell lines I'v used seem OK. The genes I'm targeting with the siRNA are all different kinds, not only essential for survival. The only condition I've changed was the brand of the agar. So, according to you analysis and suggestions, I plan to change back to previous brand and at the same time, to try the LMP agarose. I'll let you know the results afterward.

BTW, for the siRNA transfection, I've been keeping use Lipofectamine-2000. Invitrogen just proclaimed a new reagent last month, Lipofectamine RNAiMAX, which is especially for siRNA transfection. I haven't tried it yet, and I haven't tested any others. I'm wondering if you could tell me which reagent is the best after testing so many kinds of different reagents (That will be a lot of work!)

Thanks again!

Seagull
Seagull's picture
It is agar!

It is agar!

After changing back to the old brand of agar and tested again, the strange phenomenon disappeared. Anyway, thanks all of you. I've learned a lot from communicating here with you guys. I really appreciate your help!

dragonfly
dragonfly's picture
It is my first time of soft

It is my first time of soft agarose assay for colony formation. I wonder how soft/solid should the top layer of agarose be? I used 0.35% of agarose (I did not buy Seaplaque agarose, just use the agarose we used for gel electrophoresis.), and the top layer is pretty solid. I wonder how cells can grow in such a solid gel? How can cells obtain oxygen? Thank you for your reply!

marcus muench
marcus muench's picture
 0.36% is what I use for the

 0.36% is what I use for the cell layer.  

Seagull
Seagull's picture
I use 0.4% for the cell layer

I use 0.4% for the cell layer.

Greven
Greven's picture
I have a question about the

I have a question about the type of agar/agarose. It seems that I need to use a special type of agar (DNA grade). I cannot use regular agar? I tried regular agar and my cells did not grow.

Is there a reason why I must use DNA grade?

marcus muench
marcus muench's picture
 I use low melting point

 I use low melting point agarose, which is more pure than agar and easier to work with because of its melting point.  Purity is important as it reduces the likelihood of introducing something that will affect cell growth.

Greven
Greven's picture
but i autoclave my agar, then

but i autoclave my agar, then microwave it again.

marcus muench
marcus muench's picture
 I also autoclave the initial

 I also autoclave the initial preparation (briefly - 5 minutes at temp) and microwave for each use.  But that neither helps the purity nor makes it any easier to keep from scorching your cells.   

You
You's picture
Not all cancer cells can grow

Not all cancer cells can grow in Agar, you need try to find some kinds which very easy to grow.

joana
joana's picture
Hello!

Hello!
I've also had a problem with a soft agar colony. I do everything like in protocol,  0,5 % base agar ( 1 ml), 0,35 % top agarose with the cells ( 1 ml) in a 6 well plate. Everything is quite good , except feeding the cells. Do you remove off  "old:" media ?? and how to do it without getting off also the agar with cells?   After putting the new media all is liquid, without solidify.... but before feeding everything is ok, so the problem isn't in the beginnig of the test.

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Many thanks in advance

marcus muench
marcus muench's picture
 As mentioned above, I use

 As mentioned above, I use agarose-not agar.  Low melting temperature agarose, so that you don't have to add it to your cells while it is too hot.  Agarose is much more defined than agar.

You
You's picture
 

 
Yes, Agarose is better. I microwave agar, keep in 75C, mix with warmed medium. bottom 0.7%, 1hr later, middle layer with cells, 1-2hr up layer with 96 well plate. culture 2 weeks is no problem, need not change medium.

May you success.

You
You's picture
 sorry, some mis. in my

 sorry, some mis. in my previous reply.

Detail method is : 2% agar should be kept  in 45C, mixed with 37C culture medium , 0.7% for bottom layer, 50ul can cover the bottom.

top layer 0.3% with cell, can be 100ul

top 50ul medium only,  It is good for 2-3weeks.

Good luck!

joana
joana's picture
Thanks very much!

Thanks very much!

this my protocol :
Melt 1% Agar in a microwave and cool to 40°C in a waterbath (prepare in hood using autoclaved sterile
glassware).
2. Using falcon tubes, warm 2X RPMI with 20% FCS* and antibiotics to 40°C in waterbath. Allow at least
30 minutes for temperature to equilibrate. (*Serum may be replaced by agonist, a 1% FCS media or
other possible combinations depending on the experimental plans).
3. Mix equal volumes of the two solutions to give 0.5% Agar + 1X RPMI + 10% FCS.
4. Add 1.5 ml of mixture from Step #2 to each 35 mm Petri dish and set aside for 5 min. to allow agar
to solidify (These plates can be stored at 4°C for up to 1 week – let them sit at room temp for
30 min before using). See notes above for alternative volumes.

Melt 0.7% Agarose in a microwave and cool to 40°C in a waterbath (It is important not to exceed
40°C, otherwise the cells will be killed). Also warm 2X RPMI + 20% FCS (*see note for serum in
media as described above) to the same temperature.
2. Trypsinize adherent cells to release them and count the number of cells per ml. It is very helpful to
have a positive control for colony formation. Take care that a single cell suspension is obtained (see
below for example).
3. This procedure requires 5,000 cells/plate. By using 20,000/tube, there is enough to plate four agar
plates from each original tissue culture plate. Adjust the volume so that the cell count = 200,000
cells/ml.
4. Add 0.1 ml of cell suspension to 10 ml tubes.
5. Label the 35 mm base agar dishes appropriately (from Step 3). (If they have been stored, it is a
good idea to remove the plates from 4°C about 30 minutes prior to plating to allow them to
warm up to room temperature)
6. To plate, add 3 ml of 2X RPMI + 10% or 20% FCS and 3 ml 0.7% Agarose to a tube of cells from Step
4. Mix gently by swirling and add 1.5 ml to each of the three or four replicate plates. Only do one
tube at a time so that the agarose does not set prematurely.
7. Incubate plates at 37°C in humidified incubator for 10 to 30 days.
- Feed cells 1-2 times per week with cell culture media

 And my problem is only in this last senstence, during feeding..   e.g. after 1 week.    when I put media, all is liguid in a moment, not solidify. during first week  cells grow in agar, but when I feed them, all is liqiud, the cells adhere to the bottom of the plastic :/

But I'll try like you sugest.  only with agarose . I use  Low melting agarose ( Prona Reducta)

greetings from Poland!  :)

marcus muench
marcus muench's picture
 Thanks Joana for posting

 Thanks Joana for posting that detailed protocol.  

I think it would be helpful if you and others post what kind of cells they are growing in these different versions of the soft agar assay as some cells will grow under some conditions not others.

In my case, my experience with the assay is in growing murine and human hematopoietic progenitors (CFU-C).

Greetings from San Francisco

joana
joana's picture
Well, my cells are canine

Well, my cells are canine mammary gland tumor .

You
You's picture
 And my problem is only in

 And my problem is only in this last senstence, during feeding..   e.g. after 1 week.    when I put media, all is liguid in a moment, not solidify. during first week  cells grow in agar, but when I feed them, all is liqiud, the cells adhere to the bottom of the plastic 

With SeaPlaque Agarose (low melting), Lonza Rockland, Inc, cat# 50101, I have never got solification problem.      Make sure the bottom agar is 0.7% or higher, of course the high percentage will decrease visibility for plate or dish reader (the instrument can count colonies after fluorecent satin).  The cell should not invade to plastic. It is difficult to replace top medium because 0.3% agar is so soft and will be removed together with top medium. It is impossible to replace medium perfectly. IF the bottom layer become liquid, you need to increase % of agar. for big dish, and handle carefully, in case the agar separate from the bottom .

Feeding is not necessary, you can add some if you insist.

marcus muench
marcus muench's picture
 I always place the fresh

 I always place the fresh plates at 4°C for 5-10 minutes to get good solidification of the low-melting point agarose.  As stated upthread, I use 0.5% and o.36% without issue.

joana
joana's picture
Thanks a lot for your advices

Thanks a lot for your advices! 

Ludivine
Ludivine's picture
Hi,

Hi,
I'm having problems with my layer containing cells. The 0.35%(final concentration) of agarose is quite gelly and the cells have tendancy of moving  to the middle of the plate. What kind of consistency is the top cells layer suppose to have (liquid or solid)? I'm not sure if I might not be letting it set for long enough, how long should I keep it on the bench before I put it in the incubator?
Thanks

marcus muench
marcus muench's picture
 Your agarose should have an

 Your agarose should have an even consistency.  The two typical problems are that the warm agarose was added to cold medium before plating and gelled before mixing giving you an uneven mess.  The second problem is that low-melting point agarose may never really gel completely if just left to solidify at room temperature before being placed at 37°C.  I always place my plates in a refrigerator or cold room for about 5-10 minutes before placing them in the incubator.  Make sure they are solid before placing in the incubator.  Usually by the time the plates develop a little bit of condensation on the inside of the lids at 4°C they are ready to be moved.

huyenkobe
huyenkobe's picture
Seems that you are familiar

Seems that you are familiar with soft agar assay. I need your help. I am working with SeaPlaque Agarose Lonza Takara to do the soft agar assay. Base agar is 0.6% and top agar is 0.35%. My stock gel is 1.5%. According to my senior, I created the stock by adding 1.5 g of SeaPlaque Agarose to 100 ml of distilled water and autoclaved the bottle. After autoclaving, I put the bottle in 4oC and the gel solidified well. Form this stock , I created 0.6% base agar and pour the gel on 6-wells plate. After putting plate overnight at 4oC, the gel solidified well. But only after 6hrs putting at 37oC, all wells melted.Can you tell me what the problem is and how to solve it. I did it about 40 times and I was successful only 2 times. 
Here my email eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%76%68%75%79%65%6e%38%30%40%79%61%68%6f%6f%2e%63%6f%6d%22%3e%76%68%75%79%65%6e%38%30%40%79%61%68%6f%6f%2e%63%6f%6d%3c%2f%61%3e%27%29%3b')). Thank you for your help.

cloud
cloud's picture
I have encountered this

I have encountered this problem recently, is just had something to do with agar?