During MTT assay, after seeding we unknowingly forget to cover 96 well plate with the given lid and we incubated for 24hr in 5% Co2, humidified with copper sulphate.
After 24 hr of seeding, we didn’t notice any morphological changes in seeded cell.
So we continue the MTT experiment,by treating cells with interested Drug at various concentration for 24hr and allowed formazon crystal development by adding MTT at working concentration.After 4 hr incubation with MTT , we didn’t see any purple color formation in any of the well (Untreated ,treated and Positive control).
Lack of purple color indicate loss of cell viability.. Is it senescence death or other cell death like necrosis, apoptosis , What would be the cause for lack of purple color formation, is it direct effect of CO2 or copper sulphate kept for humidity .
Can cancer cell undergo cell death without morphological changes?
The description you provide about the setting of your experiments is out of my understanding.
You must wait at least 48h to draw any conclusion. I am a French speaking guy with reather (by definition) limited understanding of English wording ... But it seems to me that you are "speaking" about "24 h-generated" effects ...
If you could rephrase your comments / questions I am pretty confident that I could provide you with interesting readings ... (i.e. articles!).
Best regards
Robert
The description you provide about the setting of your experiments is out of my understanding.
You must wait at least 48h to draw any conclusion. I am a French speaking guy with reather (by definition) limited understanding of English wording ... But it seems to me that you are "speaking" about "24 h-generated" effects ...
If you could rephrase your comments / questions I am pretty confident that I could provide you with interesting readings ... (i.e. articles!).
Best regards
Robert
Hi Suresh,
MTT is mitochondrial based and a lot of things can happen with reagents, it is possible that since your negative control (i.e. killing agent) do not have a lot of signal you should consider trouble shooting the method. One quick assay to assess death economically is to use trypan blue viability exclusion as we did in our papers [(please see https://www.researchgate.net/publication/260112146_MTORp70S6K_signaling_distinguishes_routine_maintenance-level_autophagy_from_autophagic_cell_death_during_influenza_A_infection
OR http://www.sciencedirect.com/science/article/pii/S0042682214000117) OR
(http://www.nature.com/cddis/journal/v7/n3/full/cddis2015409a.html OR https://www.researchgate.net/publication/296698867_Dengue-induced_autophagy_virus_replication_and_protection_from_cell_death_require_ER_stress_PERK_pathway_activation)). This is more labor intensive than MTT but is based on membrane permeability to trypan blue. Best of luck!
-ED
Article MTOR/p70S6K signaling distinguishes routine, maintenance-lev...
Article Dengue-induced autophagy, virus replication and protection f...
correction: negative control (viable cells not killing agent). sorry, my fault!
-ED
Just a naive question, not to embarrass you. Are you sure your cells were not dried at the end of the incubation? I mean did you have enough media after the drug treatment?
Mr Emanuel
Please don't crowd a discussion forum with unnecessary citations. Trypan blue assay is a very old and common assay and for that no need to put your papers here which are irrelevant to the main question.
Ans to Suparna Sengupta ..
Yes mam, i was asked with similar question when i discussed this queries with Dr.Venil.(IIT adjuct faculty)
I noticed my well are not completely dried, it has enough media in each well.
Initially (During seeding) i filled each well of 96 plate for volume 200ul media ,unsure how much media got evaporated, but i am sure there was enough media ( more than 100ul) after 24hr incubation without lid.
Cells was not floating ,no morphological changes on the day an hour prior to drug treatment. (Visualized in 10x) ,thats why i confidently proceeded the experiment by treating with drug of interest in various concentration , untreated well and making the volume to 200ul/well.
Then on next day i did MTT .. there was no purple color development .
Why purple color failed, what happen to all my cell. Was the cell died ? or the cells would have come out prior to drug treatment ,during washing ?
If cells would have come out during washing ,before drug treatment, how adherant cell loss its contact inhibition , even after losing contact inhibition ,why it has not changed its morphology ..
Curious to see, can cancer cell die without any morphological changes .(OR) Dead cancer cell can retain intact morphology for some short period ..
As far as known so far, morphology change always happens. May be something wrong with your reagent which happens to all of us sometimes.
Mam, only after seeding the cells in 96 well plate my fellow met kept the lid open ,. I used appropriate lid to close the plate on the day of drug treatment and the following day of MTT.
plate was not kept open from the day of drug treatment ... .
Yeah! I agree with Suparna, the cells could've just dried out. A lot of the portion of the the water in the media may have evaporated leaving the solution very hypertonic leading to osmosis. The cells may have shrunk but its tough to notice.
Might be right .. Yes we might not have noticed changes via Iight microscope.
(Or)
the cell death might be intermediate between apoptosis and necrosis..
Yes i came across info (Intermediate cell death) in internet.
plz follow the below link ..
http://physiologyonline.physiology.org/content/19/3/124
Cells may end in an apoptosis-like death without activation of caspases, reflecting an alternative pathway whereby lysosomal proteases play a crucial role. In these cases condensation of chromatin is low and fragmentation of nuclei usually does not occur.
More over i suspect membrane blebbing might not have occur in this intermediate form of cell death..
It would astonishing to see a cell death without any morphological changes.. Thanks to all ..
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