Hi!  As mentioned above, FCS-containing cell culture medium contains trypsin inhibitors. It might help to rinse the cells with PBS after aspirating the cells, before adding trypsin. For highest trypsonation efficiency, warm the trypsin to 37 degrees Celsius before adding it to the cells. 

I also agree with Kersti and Elisa..I always use the following steps.. 1. First aspirate the spent medium and wash the plates with sterile PBS (appx. 5ml, pre-warmed). 2 Add 1.5-2 ml of pre-warmed trypsin. 3. Incubate at 37 degree upto 5mins or less and then tap to see the cell detachment. 4. Add FBS containing washing buffer/medium (pre-warmed)...for safety  use 10times the volume of tryspsin. 5. Pipette gently several times. That's it...For single cell morphology we routinely culture mES/iPS cells on E-cadherin based matrix (E-cadherin Fc). If your research focuses on single cell morphology you can try this  matrix.  You can find more information in the attached paper link. Thanks.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0000015

Thank you, everyone! Actually I did rinse the cells with DPBS after removing the medium, just forgot to add to my procedure in the initial ask. 

I was taught to use the medium, trpsin and other solutions incubated in a refrigerator, without pre-warm in 37 oC. I also doubted on their efficiency under a lower temperature. Can anyone give me some suggestions? Thanks a lot!

The optimum temperature of Trypsin is 37.1...so I think it's better to use that temperature for pre-warming. To avoid autolysis of trypsin it should be stored in -20 or -80 though.

I work with mammary stem cells and I use Accutase to get a single cell suspension. It is much more gentle on the cells than Trypsin. If cells are still not dissociated, I then use Trypsin 0.05%.

Do you culture your cells in 2-D or 3-D?

I also use Accutase (less aggressive than trypsin) 5 min at 37°C, followed by mechanical disaggregation, for adult human stem cells and adult mouse stem cells.

First, rinse your culture flask with phosphate buffered saline (PBS) to remove all traces of serum left behind by the aspirated medium.

Second, pre-warm your trypsin ( I recommend Life Technologies' (Gibco) TRYPLE, a genetically engineered active moiety of trypsin, without the contaminating chymotrypsin found in usual trypsin; also, it can be stored at room temperature rather than in the refrigerator) to 37 deg C.

3) Add e.g., 3 mL of the Trypsin to your culture if in a T25 flask (5 mL for a T75) and incubate at 37 deg C for 3-5 min. Check under (preferably inverted) microscope to see if cells rounded and about to detach. If so, give flask a sharp knock against your knuckles to set up a shear wave to detach the cells and separate them into a single cell suspension. If need be, incubate a bit longer and repeat process. 

4) add a volume of medium with serum equal to the trypsin volume to counteract the typsin, gently vortex.

5) take aliquot (e.g., 0.1 mL), add to equal volume of freshly filtered crystal violet solution, vortex, and count cells on a hemocytometer. For every cell you count in the 1 mm square box, you have 2x10^4 cells/mL (2 because you have a 1:1 dilution in dye solution). This gives you your cell concentration and lets you confirm that the cells are in a single cell solution. Once you have your technique reliably working, you have the option of using automated counting means, but best first to confirm that your cells are separated from each other.

6) seed an appropriate volume of your cell suspension to 5 mL (less the seed volume) for a T25 flask, or to 15 mL (less the seed volume) for a TR75 flask. Pipette up and down gently a few times and then cap your flasks and place them in your CO2 incubator at 37 deg C.

Good luck.