Optimizing Cell Suspension for Accurate Cell Counting

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Discussion Overview

The discussion revolves around optimizing the trypsinization process for HeLa cells in a biophysics laboratory setting, focusing on challenges related to cell suspension and counting accuracy. Participants share techniques, protocols, and personal experiences related to cell culture, specifically addressing issues of cell detachment and retrieval.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested
  • Experimental/applied

Main Points Raised

  • An undergraduate student reports difficulties in trypsinizing HeLa cells, noting low cell concentration and visible cells remaining on the plate.
  • Some participants suggest actively rocking the flask and tapping it to enhance cell detachment, while others recommend using a scraper for better retrieval.
  • A protocol is shared that includes washing with DPSS without Ca++, using a specific trypsin concentration, and scraping cells off the substrate.
  • There is a discussion about the appropriateness of incubating the plate during trypsinization, with differing opinions on whether this is common practice.
  • Participants express uncertainty about the effects of culture media on trypsin activity and the use of straight trypsin versus trypsin with serum.
  • Questions arise regarding the type and size of pipettes used for retrieving detached cells, with some preferring serological pipettes for their gentleness on cells.
  • Concerns are raised about the potential damage to cells when using scrapers, with suggestions to use larger pipettes for gentler handling.

Areas of Agreement / Disagreement

Participants express a variety of techniques and opinions regarding the trypsinization process, with no clear consensus on the best approach. Disagreements exist regarding the use of incubators and the effects of different trypsin formulations.

Contextual Notes

Some participants mention the importance of trypsin exposure time and concentration, as well as the role of serum in deactivating trypsin, indicating that these factors may influence the effectiveness of the cell detachment process.

Who May Find This Useful

Researchers and students involved in cell culture, particularly those working with adherent mammalian cells, may find the shared experiences and techniques relevant to their work.

moogull
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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.
 
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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.
 
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?
 
moogull said:
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.
 
gravenewworld said:
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!
 
Andy Resnick said:
serum (a culture additive) has anti-trypsin agents in it. We *never* use straight trypsin!

Yeah, I misread the post so deleted mine.
 
moogull said:
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 said:
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.
 
  • #10
Andy Resnick said:
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.
 
  • #11
moogull said:
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.