I treated MDA-MB-231 cells with my drug in suspension. I plated cells into 96 well plate after the treatment and incubated for 24 hours. I observed my samples into the microscope, and found that the cells have formed clusters and these clusters are uniformly distributed over the surface of 96 well plate. I am finding hard to understand this and don't know how to report this in a research paper.
I have attached two images, the first one is for cells treated with drug and the other one represents control.
It looks to me like your treated cells are not reattaching to the plate. Can you put the cells in your 96 well plate before treating?
Hi,
I think your cells lost their growth ability before you seed them (both treated and control cells). All floating cells look dead or they lost their attachment abilities. Dead cells just gathered, they look like colonies but I believe they are only dead.
What were your treatment conditions? How long did you treat the cells with the drug?
I agree with Cigdem, most of your cells look dead even in the untreated group. Please make sure you are using tissue culture treated plates.
Thank you all for your answers. Actually, I am interested in studying the effect of combined effect of electroporation and drug. I treat them with drug and electroporation in suspension and plate them back to study the effect after 24,48 and 72 hours. I am still to figure out the way to do electroporation on cultured cells.
Hi,
It might be by changing osmolarity with Electroboration due to cells change their shape from adherent to sphere. We have test different cells such as mesenchymal stem cells (human, rat, murine); immune cells ...You also can test viability from your cells with different concentration of drugs after shocking.
Dear Lakshya Mittal ,
To figure out the way to do electroporation on cultured cells, you can read this;
http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-67.html
Thanks for sharing the protocol swarnendra. I have been doing the same, "electroporation on the cell suspension".
Mengyu, thanks for the answer. However, the pictures attached in the answer are for non-electroporated samples: Control, and drug treatment. I was wondering about the shape of cells in drug only treatment, they look clustered. As other answers mentioned, cells seem to loose their adhesiveness. Though electroporation can effect the adhesive behavior of cells, I am not sure, how can this happen in control/drug only treatments.
Dear Lakshya
we have previously observed such changes in MDA-MB-231 cells exposed in microgravity (Masiello MG et al. Phenotypic switch induced by simulated microgravity on MDA-MB-231 breast cancer cells.). This happens as a result of several changes occurring at the cell-to-cell and cell-substratum adhesion contacts. I do not understand what kind of drug you used, but it is likely that the treatment impairs the cell-substratum interactions (perhaps by interfering with FAK or integrins or with E-cadherin-related adhesions). Yet you must check about the viability of your samples: detachment and clustering may indeed both trigger apoptotic pathways. You should take a look on pAkt/Akt, pERK/ERK as well as on cPARP. To check about adesiveness, please verify if changes occur at the vinculin level: this is a critical parameter in gasping if something happen at the cell-microenvironment level. Yet, if the drug interferes with cells adhesion property to the substratum, this is a significant outcome and could be exploited for 'therapeutic' purposes. Finally, I think that apoptosis also occurs in the treated samples, as cell density is presumably lower here than in control sample. You should made a cell count and further verify (Ki67) the growing status of both populations.
Best,
Mariano
Ussually when I am treating Cancer cells with SRC inhibitor they change the phenotype as rounded blebbing cells, it seems like they switch the type of movement to an ameoboid.
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