Hi, folks! I am currently working on MB231 culturing in 96 well plate for fluorescence study. The thing is when I wash the cells with PBS, some cells start to shrink and become a chunk; meanwhile, some cells are lost (as from microscope clearly cells become less than the time before washing). I check the research gate, people only mentioned Caco cells having the similar problem, so I am very sincere and eager to find out what is going on with the cells. My senior told me when they reach a certain confluency they become easy to shrink and flip apart from the well-plate. But my judgment is there was only 60% confluency when I was doing the washing process. So Could there be some other mysteries which are beyond my knowledge?
So basically what I did was I just gently pipetted up the medium and added in the PBS. I even tilted the well-plate to a certain angle and make sure the pipette tips never touch the cells.
Hello Kenny,
I am also using same cell line. But have never faced such issues.
I have some quick suggestions. May help you in your work.
Regards,
Utsav
Hi Kenny, apart from the useful suggestions by Utsav, If you have to use PBS, buy tissue-culture grade PBS.
MB-231s are tough adherent cells that shouldn't come off the plate unless you are using sub-optimal PBS.
PBS made in the lab can have massive quantities of endotoxin and higher or lower physiological salt concentrations which can greatly affect cell adhesion.
If you don't have to use PBS, wash the cells with serum-free media.
Hi Steing and Utvav. Now that based on your comments, I started to doubt whether my cell line is in good position. I also use Hacat (human dermal keratinocyte) for my experiment but using the same PBS to wash them never wash them away (to my understanding, Hacat is also very strongly adherent cells). That prompts me to rule out the effect of PBS (though sometimes cell-type-dependent effect would come in). But from many times of repetitive experiments, I can conclude that when they reach around 80% confluency, they tend to detach from the well plate. (means when I remove medium and wash with PBS) So, you two haven't met this 'strange' phenomenon before? Or can PBS can harm the adhesion to MB231 but not to Hacat? By the way, I really tried to wash as gently as I can....
I have the same problem with fibroblast. After washing with PBS and before irradiating many of the cells attached in the wells start to detach. Why? Could it be the PBS?
Alba Blánquez I also have the same issue with fibroblasts. Which fibroblasts do you use?
I use a primary cell of fibroblasts. I am thinking if purchasing a reference line such as 3T3 fibroblast. Which fibroblast do you use Nina Berggaard ?
I'm also using a primary cell line, from PromoCell. They are human aortic adventitial fibroblasts. I will try different types of PBS and see if it helps.
Alba Blánquez ,
I heard that PBS without calcium can affect the adhesion of cells in vitro, as Ca2+ is required for the proper function of some key proteins related to adhesion. (I forgot the names already) So in your case, is your PBS w/o calcium?
I use PBS without Ca2+. But I have already prepared a new buffer with Ca2+ or Mg2+. TGhank you very much for your advise.
Hi,
I've had the same problem of cell detachment with the MDA-MB-231after gentle PBS washing. Alba Blánquez can you tell me if your Ca2+ or Mg2+ PBS formulation overcame this problem? Thanks a lot.
Daniel
Hi Daniel,
The results obtained after whasing with PBS + Ca+Mg showed an improvement in the attachment. I lost few cells. I do not think this is the best solution but at least it worked!.
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