Optimizing Cell Suspension for Accurate Cell Counting

[moogull]

I am an undergraduate physics student working in a biophysics laboratory, and recently on the job I have started culturing cells. We need the cells in suspension for reasons I can't disclose but the important part is that I am having some difficulties in the trypsinization process.

The main problem is that when I want to count the cells. Basically, after I add the 2mL of trypsin to the plate (polystyrene I think) and let it sit for a couple minutes, I dilute the tryp with some culture medium (8mL). Then I transfer this suspension to a conic tube which is where I count from. BUT, I have noticed that the concentration is really low (around 8-16 *10^4 cells/mL) and I took a look at the plate and I can see that there appears to be a bunch of cells still on the plate! And they all seem to be bunched up near one of the edges of the plate which is probably due to the fact that I tip the plate to get all the medium up without any air. Any biologists out there that do this regularly that can give me some tips? I'm the only person in the lab that does this and they're kind of putting a lot of pressure on me ugh.

BTW, These cells are HeLa cells, the trypsin medium is TryPLE, and I do wash the cells with PBS before I trypsinize. I know for a fact that the cells are balling up when the trypsin is added, but I just can't seem to transfer them adequately.

 

[Ryan_m_b]

Whenever I've trypsinized I've not just left it to sit. Alternate between rocking the flask and firmly tapping the bottom of it. If you are still experiencing signficant adherent cells consider purchasing a scraper: http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=17216291

 

[Andy Resnick]

Our protocol for passaging cells has three main steps:

1) aspirate the media and add DPSS w/o Ca++ as a wash step. The removal of Ca++ helps promote detachment.

2) Aspirate the DPSS, add 0.05% trypsin solution and incubate at 33 C for 10 minutes. The cells should appear rounded, and some may have come off the filter.

3) Using a pipette, (gently) scrape and aspirate the cells off the substrate and place into a tube for centrifugation. The tube has a small about of serum (100 uL, and the suspended cell solution is about 1 mL) as an anti-trypsin agent.

Offhand, I'd guess you have a few problems- not enough time with the trypsin, for example. Also, if you are not physically scraping the cells off, many will remain attached.

 

[moogull]

Thank you for the suggestions, I'm going to experiment with some longer trypsin exposure times and see about getting a scraper as well. Is it common to put the plate in an incubator while waiting for the trypsin to act?

 

[moogull]

Oh, also, what size and type pipette do you all usually use when retrieving the detached cells?

 

[Ryan_m_b]

Thank you for the suggestions, I'm going to experiment with some longer trypsin exposure times and see about getting a scraper as well. Is it common to put the plate in an incubator while waiting for the trypsin to act?

Personally no, as trypsin kills cells if you are trying to do a live/dead cell count then you want to minimise the time the experiment takes. Rather than put it in an incubator just rock/bang the flask continually for about 3 minutes.

 

[Andy Resnick]

I've left my cells to sit and just waited.

Someone correct me if I'm wrong (I'm new to cell work too), but I thought culture media deactivates trypsin? Our protocol is to aspirate cells, wash 2x with PBS, then trypsinize with straight trypsin.

serum (a culture additive) has anti-trypsin agents in it. We *never* use straight trypsin!

 

[gravenewworld]

serum (a culture additive) has anti-trypsin agents in it. We *never* use straight trypsin!

Yeah, I misread the post so deleted mine.

 

[Andy Resnick]

Thank you for the suggestions, I'm going to experiment with some longer trypsin exposure times and see about getting a scraper as well. Is it common to put the plate in an incubator while waiting for the trypsin to act?

Oh, also, what size and type pipette do you all usually use when retrieving the detached cells?

I don't know about 'common'- we culture adherent mammalian epithelial cells, and most of the protocols I have seen for adherent cells have the typsination step performed at 33C. The time will depend on the trypsin concentration- for us, 0.25% is too high a concentration as the cells will get damaged if we wait even a little too long.

The pipetting is done with a 1 ml serological pipette:

http://www.fishersci.com/ecomm/servlet/fsproductdetail?storeId=10652&productId=616101&catalogId=29104&matchedCatNo=1367545&fromSearch=1&searchKey=pipette||pipet||pipettes||serological||wrapped||pipets||individually||ml||1&highlightProductsItemsFlag=Y&endecaSearchQuery=%23store%3DScientific%23nav%3D0%23rpp%3D15%23offSet%3D0%23keyWord%3D1%2Bml%2Bserological%2Bpipette%252C%2Bindividually%2Bwrapped&xrefPartType=From&savings=0.0&xrefEvent=1333673813079_0

the tip is rounded enough so the cells are not particularly damaged. Again, we are doing this as passaging, so the loss of a few cells is no big deal- YMMV.

 

[moogull]

The pipetting is done with a 1 ml serological pipette:

the tip is rounded enough so the cells are not particularly damaged. Again, we are doing this as passaging, so the loss of a few cells is no big deal- YMMV.

Does it matter a whole lot that I only have Eppendorf micropipettes? I work in another lab that does have the serological pipettes, but I doubt they would like me running around with those outside the lab.

 

[bobze]

Does it matter a whole lot that I only have Eppendorf micropipettes? I work in another lab that does have the serological pipettes, but I doubt they would like me running around with those outside the lab.

Bigger would be better. You might not have to scrap the actual cells, I would always use a .5 ml or larger pipette and gently pipette the culture medium at an angle to the cells on the plate/bottle, just pipette up and down (draw in medium, squirt out medium).

I'd try that and/or a scraper before I start doing the trypsin longer. Scrapers in my experience, can be rough on cells though--Which is why I prefer pipetting them with culture medium.