Hi,
I am working on the anticancer and proliferative effects of a plant extract. I have prepared the plant extract of a plant using different solvents. The solvents used were methanol, butanol, petroleum ether, chloroform, ethyl acetate, water, chloroform : methanol and hydroalcoholic in different ratios. The concentrations used of the extrac was 50µg/ml, 100µg/ml and 200µg/ml.
I have done MTT assay to check the anti cancer effect on A549 cells and also to check proliferative effects on PBMC. Both the results were positive but the microscopic images showed black aggregates of some sort. Are these aggregates due to killing of cells (apoptosis/necrosis) and if yes so how will it be explained in case of PBMC as the PBMC seem to be activated.
Can it be possible that these aggregates are due to the compound used (maybe because of proteins or polysaccharides. Also these aggregates were also seen in case of paclitaxel drug.
I have to synthesize nanoparticles from these extracts so if these aggregates are due to the compound then will they interfere with the formation of nanoparticles.
I am attaching images.
NOTE : Please avoid the black dots in the images as they are due to some technical faults in the microscope.
Hi Prachi, if these aggregates are floating above the cells then they are probably fungal contaminations in the cell cultures.
Thankyou Steingrimur but these aggregates are attached to the surface of the plate, so what could it possibly be ?
Hi, but anything black showing up in cell culture is not derived from the cultured cells.
Also, can you verify that the black dots in the first figure are real or not.
Hi,
The black dots are due to technical error in the microscope as mentioned earlier.
So, these black aggregates could be due to the plant extracts ?
Are those images post-MTT? They could be the formazan crystals inside the cells.
If not maybe your extracts have limited solubility to the aqueous culture medium. They might be soluble to the organic solvents you used for the extraction but then you added them to your culture medium with is all water.
Thankyou for your useful comments Evangelia . The images were taken pre - MTT and as far as the solubility is concerned I have also made extracts of water and they do no such black aggregates are observed in that case.
The extracts were also dissolved in 0.8% DMSO to prepare the stocks and for that case the extracts were soluble in it. So, is the solubility problem supposed to persist.
Do you think that this solubility issue will hinder in the synthesis process of nanoparticles ?
Dear Ms Prachi,
my guess is those are from your extract. When you extracted your sample with water you got different kind of compounds than when you extracted with the solvents you mentioned. And because you got water soluble compounds they didn't aggregate in your culture medium.
I think you should try different dilutions of your sample and see where you don't get aggregates.
For the nanoparticles, water extracts do have polyphenols that can act as reducing and stabilizing agents for your synthesis, but it depends on your plant. Additionaly, extracts from different solvents will also give you NPs with different morphology and size.
Best of luck with your experiments.
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