any methods to evaluate the attaching ability of suspension cells? - (Jan/15/2012 )

hi I am working on some kinds of leukemia cells which are normally in suspension. recently I used a lentiviral system and knocked down one gene and surprisingly found that under a low serum condition (1%FBS or less), more cells of the KD group were attaching to the bottom surface of a 12-well plate while much less cells from the control group attached. Unfortunately I didn't include a blank control without lentiviral infeciton in this experiment so I don't know whether the original cells will attach in the same condition. Anyway, even if the lentiviral infeciton will induce some change but the difference between the two groups is quite obvious. So I started to think about this observation.

Since this cell line originated from T cell leukemia so normally it doesnt have attaching ability. And also as a T cell line, migration is a basic ability of it. So the first thought coming to my mind is that knockdown of this gene might reduced the migration of this cell line and enhanced the attaching ability of it to certain kinds of matrix like ECM, although I know there is no such layer and just a normal culture plate from Nunc. Indeed, this gene has been reported to be linked to tumor invasion and in normal cells it also has a role in cell morphological development. So, is my guess reasonable?

What I really want to ask is how can I prove my hypothesis? I have thought of staining and counting the attached cells and showing the difference as a data. But I feel that would not be a convincing data, or maybe not a real data that you can put in the paper at all. So, are there other methods for testing the attachment of cells? By the way, since we already have the plan checking its migration/invasion ability by boyden chamber, so I just want to know a method to address one point, how to quantify this attachment difference?

Sorry it is a little bit too long and thank you for reading it. If you have any idea, please tell me. Thank you very much.

It sounds like your idea has some good basis. I would certainly go ahead and test the attachment. There are a few ways you could do this - my first option would be a colony forming assay - seed the cells at relatively low density and then look for the formation of colonies. The colonies can be stained by crystal violet and then all you nee to do is count the colonies.

Second would to be to do a direct assessment of the number of cells attached on the plate - probabyl by trypsinising the cells and then counting.

Third would be to do some sort of trans-well migration assay and/or an invasion (scratch) assay.

Thank you very much, bob1. Still I have several questions.

1. how do you stain the colonies since they are in the gel? actually I tried this experiment once before but failed. First I made the bottom layer with 0.6% agarose and then mixed cells in top layer with 0.3% agarose. After they got solidified, I added some medium on top to keep it from dried. That might be the reason for my failure because several days later the 0.3% layer mobilized so I couldn't count the colonies. Maybe next time I don't add extra medium.

2. I think colony formation assay assesses the anchorage-independent ability of adherent cells. Since I am dealing with T-cells which are already anchorage-independent, besides cell proliferation differences what can I get from this assay?

3. Have you ever done trans-well migration/invasion assays? I saw some videos from youtube and it seems that after cells migrate or invade to the other side of the membrane (should be 8 micron in pore size), they kind of stick to the membrane and don't fall off. So you just need to fix and stain the membrane and count the cells. What I don't understand is whether it is the same case for T-cells. Can the membrane hold T-cells? Will I lose cells during fixation/staining because there is a lot of washing?

Thank you very much.

I was assuming that you were looking at the attachment in regular cell culture plates, or perhaps gelatin coated. I don't know if this will work very well but you could perhaps stain your cells with neutral red (live cell uptake) and then seed the cells in the matrix and look for colony formation.

The crystal violet stain allows you to look for colony formation, which would be unusual in a suspension cell type such as T-cells, that way you could compare KO with normal and see if there is a quantifiable difference in attachment of what would normally be a floating cell.

I havn't actually used transwells myself, but I recall from seeing others in labs using them that you do indeed stain the filter, and being not so good at attaching you are likely to lose quite a few cells during the process, unless you are very careful with the washes. I am not sure how you would get the cells to stay attached, unless you can do some sort of crosslinking step, or keep the cells in a matrix of some sort. You could otherwise perhaps remove the cells and do some sort of cytospin or smear protocol for ICC.

Thank you very much, bob1.

I started colony formation assay again and this time I didnt add extra medium on the top layer so I suppose at least I will get a result this time, no matter good or bad. However, I didnt stain the cells because I thought even if I stained them, after 2 weeks of culture, they would lose the color anyway, am I right? So I am thinking of stain them after I can see colonies or just count them directly without staining if they are clear enough. What do you think?

you are right about the culturing plate I am using. They are uncoated normal polystyrene plate. I guess if cells become attaching to the surface, there must be certain kinds of glycoproteins expressed as those polystyrene could facilitate the binding to glycoproteins that are usually present on the cell membrane. Unfortunately, my result wasnt reproducible and now I am trying again.

About the trans-well experiment, the protocol of the kit recommends using a staining kit named Diff-Quik which seems to be able to complete from fixation to staining in 15s. Maybe in that way cell loss will be reduced. But anyway, I will keep the medium and check if there are cells in it or not, in case T-cells dont stick hard.

Thank you for all your suggestions. They are inspiring.

gyma on Sat Jan 21 04:53:26 2012 said:

You are right, neutral red wouldn't last two weeks of growth. You could count them directly - you will probably find that a white light transilluminator will help with this. You could even scan the wells on scanner that is capable of scanning slides (usually called a top light scan), which will make the counting a bit easier.

gyma on Sat Jan 21 04:53:26 2012 said:

The Diff-qick sounds OK, I was just worried about the washing steps for the usual ICC sort of proceedures, which would have washed all your cells away.

Thanks a lot. I will update it whenever I got progress.