Protocol Online logo
Top : Forum Archives: : Cell Biology

Methylene blue staining and photometry question - (Apr/18/2006 )

Hello all. I am trying to resolve an issue between myself and a lab mate..

When you are using a plate reader to monitor absorbances of methylene blue stained cells, do you read the wells dry, or do you add PBS or water to the stained cells prior to reading?

Ahhh.. the joys of moving from a plant lab to a cancer lab!

Many thanks,


I do not know the answer (sorry) but, by the way, how IS the new job, Hank?


To help you better,maybe you could add some more details on your experiment


Hi, basically I'm just doing cell proliferation assays. I plate cells on a 24- or 96-well plate at equal densities, wait a few days, aspirate off the media, and stain with methylene blue. Then, I aspirate the stain and wash before letting the plates dry. The more cells there are, the more "blue" there is. I then read these plates at 570nm at plot the absorbances. We intend to do high-throughput proliferation assays in the future, and we would like to avoid having to detach the cells from each well and count with a hemacytometer.

Basically, my question is: is it alright to read the absorbance without having liquid in the well (just dry, stained cells)?

My data seems fine (ie. tumor-suppressor mutants grow faster than wild-type cells). I can see no reason as to why this is incorrect, since I'm just interested in how much light passes through the bottom of each well. The amount of light that passes through should be proportional to the number of stained cells in the well. Moreover, I'm not looking to measure the exact number of cells; just fold-changes from well to well.

Perhaps I'm overanalyzing things here. I just want to be sure that this method is acceptable. My lab mate leaves her post-methylene blue wash in the well and then reads it. Her results aren't consistent, however.



QUOTE (aimikins @ Apr 18 2006, 02:02 PM)
I do not know the answer (sorry) but, by the way, how IS the new job, Hank?

Hey, thanks for asking!

It's going well. I had to break down and get a QA lab tech job at a food company to pay the bills. Rather than let my degree go to waste, I volunteered as a research assistant at a local university as well. So I work the QA job somewhere between 3rd and 1st shift (2am-10:30am). Afterwards, I head straight from that job to my research lab and usually work until 5 or 6pm. It's unfortunate that I am not allowed to do research on the weekends, but I suppose it gives me time to catch up on the rest that I miss during the week.

My first few weeks in the research lab were miserable. Despite reading every paper the lab published (since the early 90's!), I couldn't seem to get a grip on things. They have their own language and often forget that I am not familiar with the majority of their work (they have a ton of unpublished data). The lab is very small too... in addition to the PI, it's just me and an undergrad. The PI is on sabbatical, so she spends the majority of her time trying to piece together a few more papers from last year's data. I'm not sure if it's normal for labs to have as many side-projects as this, but it sure is confusing. Thankfully, I will be put on my own project this week. She also wants me to finish up a few experiments for an older project, so I should be getting my name on a publication or two by the time I move on (hopefully before fall).

The downside to being this busy is that I have little time to browse Bioforum sad.gif