Cytotoxicity assay - Cytotoxicity assay using methylene blue (Aug/12/2007 )
Hello...i'm an undergrad in the field of biochemistry and my final year project is regarding the cytotoxicity effects of Garcinia mangostana (mangosteen) extract on HepG2.
for the cytotoxicity assay, we'll be using methylene blue(MB) to obtain the cell number. however, i think this method is really old, and from my literature review for the past 3 weeks i still can't understand the principle of this method.
the reference that i have here from past experiments (my senior), the cells are plated in a microtiter, treated with the extract then added with glutaraldehyde--to fix LIVING cells to the wells. is this correct? how does glutaraldehyde actually fix living cells to the well?
this step is followed by the addition of MB, which wud stain the cells. i would like to know how does MB stain the cells...what does it bind to...?
from another reference...it states that MB is based on the dye inclusion method...How so? what is the property of methylene blue that makes it stain only living cells?
thank you very much for your time and i really appreciate it!
for the cytotoxicity assay, we'll be using methylene blue(MB) to obtain the cell number. however, i think this method is really old, and from my literature review for the past 3 weeks i still can't understand the principle of this method.
the reference that i have here from past experiments (my senior), the cells are plated in a microtiter, treated with the extract then added with glutaraldehyde--to fix LIVING cells to the wells. is this correct? how does glutaraldehyde actually fix living cells to the well?
this step is followed by the addition of MB, which wud stain the cells. i would like to know how does MB stain the cells...what does it bind to...?
from another reference...it states that MB is based on the dye inclusion method...How so? what is the property of methylene blue that makes it stain only living cells?
thank you very much for your time and i really appreciate it!
MB will be oxidized by living cells to a colorless product, dead cells remain blue colored; but not all dead cells are colored blue, so there remains some uncertainty;
you may use it but should perform an alternative assay such as MTT/MTS, Alamar blue, SRB assay , nucleosome-ELISA or etc in parallel
MB will be oxidized by living cells to a colorless product, dead cells remain blue colored; but not all dead cells are colored blue, so there remains some uncertainty;
you may use it but should perform an alternative assay such as MTT/MTS, Alamar blue, SRB assay , nucleosome-ELISA or etc in parallel
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Thank you for the reply! really appreciate it.
hallo aileen_lyna,
when you have access to a FACS or fluorescence microscope you could also try Annexin V Staining (for staining of apoptotic cells), Propidium Iodide (necrotic cells). That will be the simplest check of cytotoxicity. then count cells. you will know per cent of "dead" cells.
hope that helped
thank you moljul! thanks =)
currently, im'm using this reagent for my cytotoxicity assays. it's very easy and accurate but you need a multiplate reader.
http://www.promega.com/catalog/catalogprod...Viability+Assay