Hi. Try doing a quick wash of the cells with warm PBS before adding the trypsin. Plus use fresh trypsin and avoid freeze/thawing it.

Hi. Try doing a quick wash of the cells with warm PBS before adding the trypsin. Plus use fresh trypsin and avoid freeze/thawing it.

As Steingrimur suggested, washing away any residual media (containing serum that will saturate the trypsin activity) should help.

Additionally, could be you're using a different concentration of trypsin than you're used to?

Steingrimur's suggestion is good. If that doesn't work then your cells have probably been differentiating. NIH3T3 cells at high density or in certain media will terminally differentiate. I would suggest pulling an early passage and making sure you don't culture them too long and too dense in the same plate.

Also check your media. Commonly the omission of glycine or other AA can cause terminal differentiation.

As others suggest, you might want to wash cells with PBS or trypsin solution once or twice. Mg++ or Ca++ is required for attachment of cells on the surface of dishes, so make sure you are using trypsin-EDTA. Invitrogen sells trypsin with EDTA and without EDTA, both. It is also good idea to wash them with PBS containing 1mM EDTA instead of PBS.

You can also try to use trypsin 0,05% not 0,25% which most people use.

you can try higher concentration or more incubation time, previously we had this problem and we increased the incubation time to 30 min with Trypsin 1X then we used 5X for 2-3 min and it was OK.

I used a rubber mallet. A sharp blow on the bottom side helps to detach the cells.

try to use fresh Trypsin and increase the incubation time with observing the detachment under microscope

HI, pl check the following

1. Confluency of your cells. NIH-3T3 cells are fast growing and If the cells are over-confluent , then it takes time to get detatched.

2. Try doing a prewash with prewarmed sterile PBS and then leave the cells in prewarmed trypsin.

3. Do not leave the cells in trypsin for longer time. On longer incubation with trypsin, the cells get lysed.

4. Cells stop growing in the following passage.

What we generally do is to wash the cells in PBS and let the cells stay in PBS for few minutes before trypsinization. Use fresh trypsin and never freeze thaw trypsin.Its better you prepare aliquotes of trypsin in whatever your working volume and iincreasing the conc.or volume or incubation time will lead to pressureon the cells and their damage.

I would also like toknow the conc.of the trypsin you are using.because I use only 0.5 mL trypsin for a T25 flask and we buy it from Sigma using 3ml seems too much.

you can do the following:

first wash the cells with PBS @ room temperature. and let the cells stay in PBS for a few minutes.

The add trypsin and make sure it covers the complete surface.

incubate at 37 degrees C for 5 mins (max 8 mins). u can look for the cell lift off.

Immediately add serum containing medium in double the volume of trypsin (i.e if u added 0.5 ml trypsin, add 1 ml serum medium) to stop the trypsin activity. otherwise cells will get killed.

now flush gently the cells from all sides of the flask. collect the suspension and centrifuge and harvest the cells.

And also please remember to wash the cells thorougly with PBS before trypsinization to remove residual serum medium

try adding some chelating agent like EDTA. it will bind the divalent ions which are helpful in cell attachment to the surface. try using it hope it works out for u...

store trypsin in 4C, do not freeze-thaw

My advice is in agreement with many of those who already replied to you:

1. try to harvest your cells before they are fully confluent

2. we usually aliquot trypsin in small volumes and do not freeze-thaw more than once (thaw-use-discard)

3. first wash with PBS at r.t. or 37ºC with gentle shaking, discard the PBS

4. make sure the surface of the culture flask is covered with a "film" of trypsin

5. let trypsin act at 37ºC for ~5 minutes, sometimes you may need to knock the flask gently on the palm of your hand to help

6. check on the microscope before harvesting

7. wash with pbs

As an altrenative use a gentle cell scraper! With small numbers of flasks I personally prefer the scraper method (but still don´t forget to check under the microscope).

If you still have problem, then try to give a wash with trypsin solution itself (2-3ml) before incubating with fresh trypsin ( I assume you are using standard trypsin (0.05%) EDTA). Confluent fibroblasts are difficult to detach and will need longer trypsinisation. Normally they are sturdy cells and are not much sensitive to longer trypsin digestion. I prefer longer time than tapping in between for fibroblasts, since tapping in between can lead to large cell clumps, which are difficult to count ( and many cells in the bunches will die after replating).

1.check your trypsin because maybe it expired or freezed for more than one time.

2.check the measure of your incubatore.

3.wash your flask with PBS before adding trypsin.

4. pipeting after incubation but remember after incubation add PBS equal volume to the trypsin that you had add before.

I think pipeting can solve your problem

Use cell scrapper to detach the cells if your are not using it....

Try to wash the cells before incubation whit trypsin whit PBS and incubating for ten minute at 37°C, and added the EDTA ( 0.02%) to the trypsin.( 0.05%).

Hi! A lot of people gave you good tips already, about washing with PBS and using a chelating agent such as EDTA together with trypsin. Also, check the concentration of the trypsin. When I was at UCSF I used a 0.25% trypsin solution. It is a high concentration, that makes the cells to be released but you have to keep an eye under the inverted microscope because it works very fast. Make sure that you warm the PBS and the trypsin at 37ºC before using, what will also help trypsin activity. What I personally do is to tap or slam the bottom of the flask to help to release the cells when tre trypsin film if there. I had tried to use a scraper, but this makes you loose roughly 50% of your cells, so I don't like it very much...

you should be sure that cell culture medium is completely washed away with PBS before using trypsin; moreover, if you are used to store trypsin at 4° after thawing, please note that trypsin efficiency decreases day by day, so you have to increase trypsin volumes. some "soft mechanical shock" (gently) on the sides of the flask could also help to detach.

1) before trypsinisation wash your cells thoroughly, because serum may deactivate trypsin

2) different cells need different trypsinisation timing. Few could be easily harvested and some other cells need more time to detach. So prolong the incubation time

3)check the quality of trypsin.try to use fresh reagent. You can add some indicators to trypsin solution to confirm its fresh or not

4) gently tapping the lower surface of flask (even though not much advisable) also could help in detaching cells

Try these:

1. Warm up trypsin in water bath before use.

2. Add trypsin and leave in incubator for 15-30 minutes (or longer until cells are observed to detach)

3. Some tissue culture media has calcium/magnesium (and these inhibit trypsin), wash cells with EDTA (divalent cation chelator) before applying trypsin may help.

I work with some 'stubborn' cell lines from time to time and I try different methods, as mentioned above, without using cell scrapper (to maintain cell integrity).

Hope you find a solution!

Wash the cells in PBS before adding trypsin. The serum that you use in culture medium has several inhibitors of trypsin and so some cell lines are not easily detached in the presence of trypsin. Washing first with PBS helps- Incubation at 37C for 5 minutes with trypsin is also advisable.

Lots of good tips above, also depending on how many times you have trypsinised in the past and if you have left the trypsin on for a bit longer, then over time some cell lines can get more resistant to the trypsin and take longer to come free and so replacing the cell line maybe warranted if this is the case.

Key items I have found in the past is:

1) not spreading yourself too thinly I.e. focus on the task at hand, it's surprising how many people are thinking about the next thing they need to do when doing cell culture

2) remove all remnants of sera as suggested above

3) check your concentration of trypsin, perhaps running optimisation experiments

4) use small aliquots as other have suggested so you are not continually freeze thawing the trypsin

5) check expiry date

6) note that sera used can vary significantly from lot to lot even from the same supplier and this can have a knockon affect on multiple thimgs including the assays and the sensitivity of cells to trypsin over time. Have a look at this article which covers a lot of the salient summary points with references relating to the effect of sera on cells from GIBCO (BioTechniques, Vol. 42, No. 3, March 2007, pp. 382–384).

7) make sure the tyrpsin is at the right temp when added

8) to avoid issues with some cells becoming more resistant to trypsin or just generally reducing risk of running out of cells when they get irreparably damaged then one thing i am a fan of is creating your bank to use- e.g. get in 3 seed vials from an appropriate company, then store 2, use the other to create a master stock of vials to freeze down with minimal passages, then create a working bank from one of the master vials, freeze down the working bank aliquots. Then only use your working bank vials for specific durations and minimise the passages, once you have used up a working bank then take another master bank vial and create a new working bank. At each step check the cells are fit for purpose too. This may not be possible due to cost and time or may not be suitable for your specific cell line though.

Good luck and all the best

Lastly ATCC provide your core instructions for the cells you using for subculturing at the following site but you probably already have this info so may not help more:

http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=CRL-1658&Template=cellBiology

1-Usually trypsine might be deactivated due to the presence of foetus bovine serum (FBS) in the growth media. Thus, you need to wash many times by phosphate buffered saline (PBS) before trypsination.

2- Try to use fresh trypsine because repeated freezing/thawing precess can reduce it trypsine activity.

3- Moreover, I would suggest to use Accutase® (a combination of protease and collagen in PBS with 0.5mM EDTA) in stead of trypsine, because it is powerful and less sensitive to freeze/thawing process.

The first and most important thing is to wash the culture plate with 1X PBS to remove all serum and remainings. Then add trypsing 1X, 1ml incubate for 2min in the CO2 incubator (if you feel necessay), then tap the plate in the bottom. PBS wash is important, if you did not wash then it would be difficult. If you even detached them by some force they will form clums.

Let us know your progress.

Aliquote tripsin, freezing thawing is not good.

you can wash the culture two times with Trypsin and the third time kip the culture 2 to 3 minutes at 37°C incubator and then tap the flask. for these culture you can close the flask after one day of incubation in the co2 incubator to decrease the cells attachment

I have the same problem with MDCK cells - known to be resistent to trypsin. Normally I wash the cells twice with PBS: the first time very quickly just to remove medium/serum (that inactivates trypsin) and the second time I incubate them at 37oC with PBS for 10-15 minutes (or even longer, just take a look to be sure that the cells are not detaching). Than I use trypsin: 2-3 min incubation at 37oC.

You can also get specific information about subculturing in ATCC.

In our lab we use Tryple ( http://es-ar.invitrogen.com/site/mx/es/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/reagents/Trypsin/TrypLE-Express.html ). It's more gentle with my cell (Caco, A549 also HEK).

Are you sure you have washed cells before trypsinize them? The serum in the culture medium will deactivate trypsin so that they are hard to trypsinize.

3 ml for 25cm2 flask is well enough. In fact, we use 0.5ml trypsin to do that...

It also depends on the activity of trypsin you are using. How many units/ml have your stock? If it's diluted it's more probable that serum remnants inhibit the enzyme.

Dear Dana,

I agree with many of the responses you have received thus far.

There are a number of things (in general) you need to keep in mind when trying to trypsinize cells.

1) Alaways view the cells prior to trypsinization to make sure that the cells look healthy.

2) Always wash the cells with PBS (one that is at room temperature is fine, does not have to be at 37C) after you have removed the growth medium. For a 25cm2, add about 3mls and give give the flask a very gentle swirl to wash all the FBS from the surface. Alternatively, after adding the PBS you can leave the flask (with the lid on!!) resting flat for about 1-2 minutes.

3) Always use fresh Trypsin/EDTA. I suggest that you aliquot the bottle of Trypsin/EDTA into small vials (5ml tubes) of 4mls per tube, which you can use as and when required. Once thawed, any quantity of trypsin/EDTA that remains, DO NOT store it in the fridge, simply discard it. The chances are you will either forget about it and when you do remember it, it will be no longer be active (see point 4 below), or someone else wil open it by mistake (which may lead to contamination). Therefore, just throw it away.

4) Do not use excessive amouts of Trypsin. You are using 3mls/25cm2 is far too high. you may actually end up damaging the cells. I know different cell types may need different amounts, but as a rule of thumb, based on the protocols given by ECACC and sigma catalogues, I tell students to use 0.5ml/25cm2, 2.0mls/75cm2 and 4mls/175cm2. You need just enough to cover the surface of the flask.

If you are using Trypsin/EDTA which you have stored in the fridge for a long time, then probably its activity has diminished, therefore, throw it away (see point 3 above for storage).

5) Make sure that you DO NOT leave the cells exposed to the Trypsin for a long time. If they start to look rounded, then they are ready, you just need to give them a gentle tap with your hands on the side of the flask, that will help them to detach fully. Try it as a practice run on a flask of unwanted cells.

6) Finally, make it a routine to do a cell count after you have harvested and re-suspended the cells in fresh medium. Do the counting using Dye-exclusion test with Trypan Blue. You should be looking at a percentage of live cells in the regions of 85-95% (at least), if your harvesting treatment is gentle and efficient. So this is a good way to help optimize your treatment of the cells, and to make sure that they have not been damaged by excesive amount and/or exposure to trypsin, as well as excessive centrifugation. Once you are confident of your treatment, then you can stop doing the cell counting.

All will be well. Have faith - hang in there.

With Best Wishes

Basil

I agree most of suggestion. To wash away serum is important. But I want to mention that not to use the PBS with Ca+2 and Mg2+, Only use PBS without Ca+2 and Mg+2. Because Ca+2 and Mg2+ will chelate with EDTA so that Trypsin/EDTA activity become lower. Another thing is only rinse cells with trypsin, then incubate cells with trypsin at 37, co2 incubator for at least 5 min . remember don't pat the flask before incubation, which interrupt the trypsin action.

Well its a very small problem. I too faced the same while working on HT29 cells. All you need to do that use 1X PBS which is sterile and completely free from Ca2+ and Mg2+ ions. Wash the flask twice (5ml) to remove all traces of serum. Dilute the 10X Trypsin-EDTA stock with either PBS or plane media (w/o serum) to 1X. 1 ml of such 1X Trypsin-EDTA is enough for 25cm2 T-flask. Keep the T-flask for 5-10 mins in CO2 incubator and try to check the cells with very gentle spin (horizontally) by hand. It will also evenly disperse the solution every time. Because some times the level of CO2 incubator shelf is not always horizontal so may be most of the solution used to gather at one place leaving other cell untouched. You may also check cells in T-flask under the inverted light microscope after 5 mins (after 2-3 horizontal spin by hands). If most of the cells are not floating then you may keep the flask again in CO2 incubator for another 5 mins and may repeat the spins.

Try this and I hope you would surely recover maximum cells. Good luck.

I agree to most of the suggestion here.

Detachment of cells depend on the adhesion property of cell, and thus trypsinisation method and time may vary with cell type.

It was not clear whether you use Trypsin only or Trypsin EDTA to detach 3T3 cell. I would use Trypsin/EDTA to detach the cells. I have previously worked with 3T3 cells, and they do grow quite fast. Never allow culture to become 90-100 confluent, this which will take more time and more trypsin to detach the cells.

I normally remove medium, rinse the flask with serum-free medium or PBS. Then rinse with with 0.25% trypsin/0.5mM EDTA solution. Remove the Trypsin/EDTA solution, and add an additional 1 to 2 ml of trypsin-EDTA solution. Allow the flask to sit at 37C until the cells detach. This is usually 3-5 minutes. You can monitor the detachment for first time. After 3minutes tap the flask and if all the cells have detached Add fresh culture medium with serum to inactivate Trypsin, aspirate and proceed with subculture or storage. or you can after tapping leave it for another 2 minutes.

I hope it help.

I remove medium, rinse rith PBS and then rinse with 0.5 ml of 0,25% trypsin (flask T25), at 37 C for 30 s, with shaking slowy. I use Vero cell.

prolonged cell culture may cause strong attachment to culture flask which is mediated partly by calcium and cadherines. pure trypsin fails to detach and may need to add EDTA to chellate calcium ion

What is also important, is the plastic type of the flask. Try another brand. Besides never harvest cells close to confluence because isolated mitochondria have shown decreased by 20-30 mV membrane potential (my own data), which is the sign of apoptosis. 60-70% maximum. Avoid over exposure to tripsin. Therefore look for another brand of plastic.

I too agree with the suggestions here and thanks to any I picked up tips from. We wash our cells with serum free media prior to trypsinising. In the past I have also used sterile PBS but could not tell you anyything about the Mg and Ca presence.

Hi, you may try TrypLe from invitrogen. I never had problem with that. All my cells detach after 10 mins incubation. Good Luck

I agree with the suggestions above but just like to add that pre-warming the Trypsin/EDTA to 37 degrees Celsius helps a lot for MDCK cells.

Try to rinse the cells with EDTA before trypsinization

Hi,

If you incubate longer time this might harm the cell membrane, so it is better to incubate for short time with trypsin. There are few things to consider

1) Ca2+ and Mg2+ free buffer for wash out.

2) Concentration of Trypsin

3) Temperature

4) Time

5) EDTA

If you use higer concentration of trypsin for longer time you will have small number of cell. Also this will cause defective cell membrane.

You can try with this protocol I got from Scott Johnstone. That worked for me, I hope this also will work for you.

Here is how I would do it:

- remove media from flask

- wash cells in flask x2 in warm PBS /HBSS (-Ca, -mg)- to remove all calcium from media that can inactivate the trypsin. (remove all excess PBS

- add trypsin (1ml of 10x, not diluted) and incubate for about 1-2 minutes in incubator and check under scope for detachment. It reduces the time in trypsin. I have tried diluted trypsin but it takes longer and the cells don't fare as well.

- I would then immediately stop trypsin buy adding fresh media (9ml, don't remove the trypsin) and remove the contents of the flask to a sterile tube i.e. 15ml

- I would count cells quickly, (as some cells settle and adhere quickly)

Wash cells with sterile PBS, trypsinize for no more then 5 min at 37, then use a cell scraper.

The buffer exchange or wash might do the trick. Trypsin is pretty robust but in the end it needs to be in a pH environment above 7 which you should get if you wash with PBS first. Also, if your trypsin has gone through several freeze/thaw events, it may have lost activity.

Hi Dana

I think you should try other way to dettach your cells, you can try the ET-buffer (10 mM Tris + 2 mM EDTA) at pH 7.8, this work very fine for me...But of course, we need to know what do you intend to do with the cells, after the dettaching...In my case, i was looking for the membranes, so i had no problem with hypotonic buffers, because I was disrupting the cells anyway with an potter homogenizer...In some cases, EDTA treatment is more effective than trypsinization...Some hits (mechanical strokes) on the side of the plastic bottle also helps when the cells are dettaching with EDTA...It works flawlessly with my Vero, CACO-2 and BHK cells...Good luck

I second the physical agitation mentioned by Fontes above. For example, in a 25 square cm flask I add 2ml trypsin and rock the flask gently for 1 to 5 minutes while observing cells under a microscope. When I see that most cells have begun to ball up, I remove ~1.5ml of the trypsin and replace the cap tightly. Then, while holding the flask horizontal in one hand, I clap the flask abruptly against my other hand perhaps twenty times and then check for detachment under the scope again. I repeat this as neecessary until it appears all cells are detached, then neutralize the trypsin immediately with serum-containing resuspension media.

Hi,

I am agree with Steingrimur's suggestion. I want to add another point...after washing with PBS, wash the flask again with 1 ml of trypsin, discurd it and again add 2 ml of fresh trypsin, then incubate at 37 temperature for 2 to 5 minutes.

Hi,

Try Accutase. It works quite good.

All of the above suggestions are reasonable: your trypsin stock might have been too diluted, or thawn too many times; also, washing the clls twice is important, to get rid of residual serum antitrypsin; usually these are the two main reasons for failure.

Dana,

The NIH 3T3 cells will form their own substrate for cell growth. The substrate is composed predominantly of type-I collagen. Therefore, I would suggest that instead of using trypsin, which will cleve proteins between lysine and arginine residues (i.e., cell membranes and thus damage your cells) you try a type-I collagenase (Worthington) instead. Also, the cells attach to their substrate via two separate binding sites: an RGD binding site and a Ca+2-cofactor binding site. So, best to dissolve the collagenase enzyme in EGTA (specific calcium chelator) -containing Ca+2-free/Mg+2-free DPBS. Protocol would be to pur off medium and incubate twice for 5 min with 3 x medium volume of ambient temperature Ca+2/Mg+2-containing DPBS to wash off any non-specifically bound material. Next, incubate for 5 min with ambient temperature EGTA containing Ca+2-free/Mg+2-free DPBS. Since EGTA has a higher binding coefficient than the Ca-binding site, this will remove the calcium from the binding site and the cells will begin to round up. Next, incubate the culture for 30-60 sec with the ambient temperature collagenase enzyme (store collagenase in refrigerator at 4C, allow to stand to warm up to ambient temperature before use and make enough to last about 3 months. Thus quantity will vary depending on useage). If the cells are in a confluent layer then the cells will lift off as sheets of cells. To get the sheets of cells into suspension you will need to triturate the cell-enzyme solution. You can negate the effects of the enzyme by neutralizing with a sterile 1% collagen solution (1:100 w/v in Ca+2/Mg+2-containing DPBS) [Easiest to make up is by putting 1 g of gelatin (type-I collagen) in 100 mls of Ca+2/Mg+2-containing DPBS and autoclaving on slow exhaust cycle. Wait until solution (glass container) is body temp (37C) to ambient temp (22-25C) before adding cells (hence, make solution up ahead of time and store at ambient temp).] Add cell suspension to 2-ml of 1% collagen solution in 15-ml polypropylene tubes (cells will not stick to polypropylene tubes, whereas they will stick to polystyrene tubes). Once cells added, screw cap on tube, invert 3-4 times to mix and then centrifuge at appropriate speed to pellet your cells. [You will need to run a speed response curve to determine optimum speed for pelleting cells - too slow and percentage of cells still in suspension, too fast and you risk killing cells (shear forces). Easy to check with hemocytometer and 0.4% Trypan blue. Use 20 microlites of potential cells (either diluted cell pellet or supernatant) + 20 microliters trypan blue, triturate to mix contents and then place on hemocytometer. Live cells will glow like headlights; dead cells will be spherical and blue in color; and debris will be blue with sharp edges. If your speed is correct, > 95% of the cells in the pellet should glow like headlights and the remaining < 5% of the cells should be either dark blue spheres or blue pieces with sharp edges, and there should be NO cells in the supernatant.] Each cell line / phenotype derivative that you use will have a slightly different pelleting speed. Once you know the speed for each line, it just becomes a matter of cook-book recipes to process your cells or derivatives of your cells.

Hi, try doing a quick wash cells (37°C) with trypsin (37°C) and then incubate with fresh trypsin (not longer than 5 min).

If your previous protocol was working, I suspect the trypsin reagent is bad. Call the manufacturer, tell them the problem you're having, and ask them if there have been similar reports for this lot number. The two times I've done this with Cellgro, the company's responses have been "YES, other investigators have reported similar issues, we'll be overnighting a replacement batch to you immediately".

I would agree with Katherine, your protocol was working before, so I would suspect your trypsin has lost its activity. Is anyone else in your lab using it either on 3T3s or better still, on another cell line?

In addition, once we defrost a 50ml aliquot of trypsin-EDTA, we keep it at 4C until it is finished, usually around 6-8 weeks. We don't refreeze, and it seems to keep its activity over this time period pretty well.

Good luck!

I also recommend Accutase(TM) as suggested by Clifford Nwaeburu. It contains mixed enzymatic activities and works for many cell types.

try using a PBS whithout calcium and cloridium..whith C2C12 cells if i use the usual PBS they don't detach!!!

PBS wash removes protease inhibitors that can neutralize the ability of trypsin to protealyse the arginine and lysine residues on the ECAMs. If you don't wash you get a very reduced trysinzation. Also, 2 commonly used percentages of trypsin are 0.05 and 0.25%. I would recommend the later (0.25%) trypsin as 0.05 is often time not sufficient to induce trypsinization in cell lines that are very sticky (ie. Vero, HeLa, 293, etc..), whereas 0.05% is sufficient for U2OS and other "less sticky" cell llines.

Trypsin undergoes autocatalysis when kept at 37 deg C, that can make it inactive. Serum contains a1 anti-trypsin, if not washed properly could inactivate trypsin. You could probably go for 0.25% trypsin, if using 0.05%. Make sure you are using Ca, Mg free Trypsin EDTA. You could probably rinse with little trypsin and then add fresh and incubate.

Hi Dana this is acvery strange problem, usually this should not happen. trypsin-EDTA detaches the cells very efficiently. I would suggest to prepare trypsin-EDTA solution of the concentration o.25%trypsin and 0.02%EDTA in cCa and Mg free PBS and make 50 ml aliquots. The problem faced by you might be due to the reduce activity of the detaching solution because of multiple exposure to room temp. and cold storage.

Follow this protocol: Discard the media completely followed by washing the cell monolayer with 2-3 ml of Trypsin-EDTA solution for few seconds and discard. This step is to remove the traces of FBS which is actually the inhibitor of Trypsin. After u hav washed the the cell monolayer. After this add trypsin -EDTA solution ( i generally use 1ml for T25 flask and rotate it properly to spread the trypsin, u can use 2 ml even ) keep it for few minutes with continuous monitoring under microscope generally this cell line detached after 2 min but please monitor. Once u can see that the cells are detached deactivate the trypsin with FBS proceed for ur normal procedure.

Good luck

Try to wash the flask with PBS then add trypsin and versen (EDTA) 1:1 for few second with continuous agitation until the cells start to detached. However if your culture is old i.e. the cell remain in the same flask for more than 10 days you'll expect such problem. Try to use fresh culture.

After u take out the cell from incubator then giving some mechanical knock, the cell will flow on the media.

I wash my flask twice with EDTA, this makes the trypsin act much faster. Also, I have found that if your cells are "super-confluent" they will be harder to detach.

Dana, as Tiwari said above, this is a strange problem. NIH3T3 cells are one of the loosely adherent cells (similar to HEK293T) that can be simply flushed out from the plate--without trypsin! The fact that yours got so sticky raises the fear that they are no longer NIH3T3, but have been replaced by a sticky, contaminating cell line. Is this problem new, or are you facing this problem with NIH3T3 cells ever since you handled them? Do you use the same medium for multiple cell lines, and do you have any stickier-than-normal-NIH3T3 cells growing? Like, a non-tumorigenic mammary epithelial line? I suggest you follow previous suggestions of using fresh media, fresh trypsin, a trypsin wash etc., but also I suggest you begin a fresh line. Get a vial fresh from liqN2 and start with all fresh ingredients. Everything anew. Killing a few days for assured success is, any day, better than killing weeks aimlessly.

Try to wash with Trypsin/EDTA solution first and then add new fresh trypsin versin solution and leave for 5 minute in 37c and it will be ok

Here is the protocol from ATCC for your cell. Enjoy:)

Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

1.Remove and discard culture medium.

2.Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

3.Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.

4.Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.

5.Add appropriate aliquots of the cell suspension to new culture vessels.

6.Incubate cultures at 37°C.

DO NOT ALLOW THE CELLS TO BECOME CONFLUENT Subculture at least twice per week at 80% confluence or less.

Subcultivation Ratio: Inoculate 3 to 5 X 10^3 cells/cm2

Medium Renewal: Twice per week

http://www.atcc.org/Products/All/CRL-1658.aspx#7301B7F956944F8382B6192957C08A3B

Check your Trypsin-EDTA solution, may be it has been deactivated. I use 5 fold diluted concentration than ATCC recommended levels and still works fine for NIH 3T3 cells.

Ya 1ml of trypsin edta 100x 1:4 dilution with pbs 1x works better for cell detachment ,before putting trypsin,you should wash with 1xpbs at least three times. And check your trypsin.

You can put trypsin and put in incubator at 37 degree for few seconds ,check as soon as cells become round in shape,you can take out trypsin and detach the cells.

This dilution was for t-25 flask

Dear Dana,

Just use 1 ml EDTA to wash cells and then 0,5 ml of % 0,25 trypsin. Expire date of EDTA and trypsin is important besides the passage number of the culture. On the other hand, viability should be more than % 85.

Wash cell cultures with PBS to remove serum which deactivated trypsin prior to trypsinize the cells.

Washing with PBS is an essential step to avoid to inhibit Trypsin enzyme, yuo can also extent the time of enzyme incubatio at 37°C and as already suggested avoid the cells reach a confluence >80%

Hi, Akilbecova. I really think you should provide feedback to us so that we won't receive e-mail alerts without your solution.

Actually I am interested in whether you have washed cells before applying trypsin detachment. If the answer is yes, that would be very interesting.

I believe most of us were very glad to see how you have solved this problems.

If this is a recent problem, I guess you do wash your cells well with PBS before trypsinising. If your cells normally come off fine, it suggests a change in the cells (assuming your trypsin/EDTA is fresh and has not been through many freeze thaws). May be worth trying collagenase to digest the ECM the cells produce. However, even more importantly, if this really is a change in cell behaviour (and not a change in method or reagents no longer working), then this is very important, because the impact on the behaviour of your cells to reagents you are using in your actual experiments may also change. Good luck.

Hi, we always wash the cells 2x with HEPES or PBS/EDTA solution to remove any residual FBS that inactivates the Trypsin. Then, add 1nl of Trypsin/EDTA for a T25 flask or 3ml for a T75 and inclubate in the incubator for 2-4min. check under the microscope for detachment and if there are still attched cells we tap the flask on the side to aid detachment. Add fresh culture medium with FBS to inactivate the Trypsin and you are good to go. I hope that helps.

Hi, I had this problem with some sort of cells but I hit the lateral borders of flask with my hand 5 or 6 times after trypsin usage tightly. It was useful for cell detachment. Trypsin is toxic . It should not be used more than 3 to 4 minutes.

I wondered how confluent the cells are. If you grow cells past, say 60% confluence, they become much harder to remove.

Obviously, try it with fresh trypsin/EDTA to eliminate the possibility that the solution is too old. Here is a tip: I like to rinse the cells with a small amount of 37C trypsin/EDTA (like 1 ml here) instead of PBS or other buffers. First, it is one less reagent involved in the process (lower risk). Second, it is quicker (about 15 s or so). Third, you can save a pipette (save the earth!). Finally, it actually inactivates the trypsin inhibitors form the serum and binds the free calcium ions instead of simply diluting them ( I am summing you meant Trypsin/EDTA, not just trypsin).

If the cells have been confluent for a while, or if the media contains ascorbate salts, or both, then your problem is collagen secretion as Henry Young above. However, I believe that collagenase requires Ca++ as a cofactor so I would advise against using EGTA or EDTA with it. This type of treatment will not be give you the usual cell suspension and more likely significant clumps. This will depend on the incubation time and the type of collagenase you use. As mentioned by Claire Stewart, the cells can behave differently after this treatment. For one, they may be in large clumps and have to migrate out of the clumps to proliferate (like in explant primary cultures). Also, collagenase often contain various levels of contaminating enzymatic activities that affect cell recovery. Overall, you recovery efficiency will be adversely affected and the experiment you plan to use the cells for can likely be affected. I would recommend passing the cells one more time before using them, if not start anew. The solution here is simple don't let the cells sit for too long and avoid ascorbate salts in the media

try to wash with warm PBS for 20-30 sec. then trypsinize. you may increase the incubation time and if not detouched then tap the flask slightly harder from both side.

Use fresh trypsin prewarmed at 37

Before u apply trypsin, please wash with HBSS three time then deposit 1ml EDTA-Trypsin (better for dissociate) and incubate for 5 minutes. After incubation, apply some mechanical knock to deattach the cells.

Is there a reason why you couldn't just use a cell scraper?

Wash with prewarmed DPBS and change your trypsin lot.

Yesterday I made cell line out of lung cancer derived from patient. I put it through the 40 um filter to provide single cells. I finished about 8 pm. Today morning I came to laboratory and I wanted to put it onto CytoSelect Clonogenic Tumor Cell Isolation

Kit. Unfortunately some of my cells already attached to the bottle (after like 12h). So I used TrypLE Express to detach them (before washed with warm PBS). It usually takes like 3 min, but after 20 min of incubation and knocking the bottle they were still hardly attached. I deactivated and removed TryplE Express and put ordinary trypsin inside. After another 20 min they still didn't react so my only way was to scratch them from the bottle. 

It is the first time something like that happened. I have never cultured lung cancer and I have no idea if it's typical for this type of cancer. I have no idea what to do if it will keep happenning.

Dana,

If you cultured the cells with ascorbate and/or if you let them stay confluent for a long time, the problem is likely collagen related as explained by Henry.  If that's not the case, then your trypsin is probably to blame (especially if that's something that you have been doing routinely) .  As mentioned above, use a fresh batch, use it warmed, incubate warm (maybe longer) and rise well before hand ( I like to use a little trypsin to rinse). Let us know what worked!

Struggling with this problem at the moment, I've always found that a trypsin wash is effective in helping detach particularly sticky cells.

Hi, i have been working with BM and  primary breast tumours and osteosarcomas and for stubborn cells, I always wash them first a couple of times with pre-37C-warmed HEPES and then a couple of times with warm versene-EDTA solution. Then I trypsinise i.e. 3ml for a T75 flask for 2-4min. Sometimes tapping the flask by hand for some mechanical aid also helps. There are cases though that to reduce the chance of over-trypsinisation and killing of the cells you could try a cell scraper although not recommended for routine use as also disrupts the cells.

good luck

Hi, just wonder can we mix TrypLE express (0.05% Trypsin 0.53 mM EDTA) and normal Trypsin-EDTA (EDTA (0.25% Trypsin, 1 mM EDTA.4Na), for example 1:1 ratio?   SinceTrypLe is hard to detach cells...