Hi,

Instead of trypsinizing cells, you could try scraping them in PBS or even straight in DPBS. This would save at least one centrifugation step and viability of the cells should be better. However, some cell lines are very sensitive to scraping, but you could try this.

Anne Höytö, i am afraid that scraping will increase cell death. Anyhow it is an extra option. Thank you.

Hello Salman,

Yes, scraping the cells would increase the cell death. There are some problems with your protocol. There are too much centrifugation steps and it will evantually lead to cell death. You are using trypsin,then centrifuge- PBS then centrifuge and then redisperse in DPBS ..ohh..

Seriously dear, the main cause of cell death during these type of experiments are only two. First, harsh pipetting while retropipetting too much. Second is excessive centrifugation.

Cells are like our babies so handle them like them.. Gentle and with love !

So here is the soltuion- After trypsinization use a slow centrifugation like 2000rpm for only 2 minutes if the cell no. is low, you can go for 3,000rpm for 4-5 min. Then resuspend the cells in PBS. Take care to resuspend then gently using a 200ul pipette tip and not with 1ml and your pipette tip should always remain in solution while making suspension. Don't force the cslls in eppendorf. Then once you repeat a gentle centrifugation step discard the PBS in supernatant but tc that the eppendorf retains some 20-50ul of PBS. Now resuspend the cells to single cell suspension in this PBS only and gently add 90% ethanol along the wells drop by drop and this rocess needs to be really very slow as if you end up with clusters of cells due to sudden addition of ethanol then you will not be able to redissociate it, doesn't matter how much reropipetting you do. (most crucial and patient step).

Please have a look:

1. Prolonged exposure to trypsin might damage cell membranes and cause cell loss. Melican D, et al., 2005. Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer. Theriogenology, 63: 1549–1563.

2. Over-trypsinization is a common cause of subculture problem which can damage the cell surface by digesting exposed cell surface proteins.

Glade CP, et al., 1996. Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol. Arch Dermatol Res, 288: 203-210.

That totally depends on the cell type you are working with....you should understand the cells you are dealing with first... In my experience with human retinal endothelial cells, they are very sensitive and i tend to wash the media and used Gibco® Trypsin/EDTA 0.025 for 5-8 min incubational time... then, collect them in 50ml tube "wider better" and added 1:1 volume full medium.. and centrifugate 1000X rpm once. try to wash with full medium twice before dispersed the cell again using large orifice pipette and it works so well. 

Good luck 

1. To avoid over-trypsinization, you may need add less volume of trypsin into cells and observe the cell morphology under microscope. Once the cells become bright and a bit round, the trypisinization process should be terminated by medium with FBS. 

2. The speed of centrifigation should be under 1500rpm, even 1000rpm for 5 minutes. 

Hope it help. 

Keeping the tubes in ice water to achieve 4 degrees C (not merely "on ice" which achieves ~ 7 degrees C) will help slow the enzymatic rate ot caspases that are undesirably activated by detachment.   And use a temperature-controlled centrifuge set to 4 degrees C.   Resuspend pellets by gentle pipetting (never vortex).   Finally, Vindelov's PI solution allows fixation and permeabilization plus PI all in one step, reducing the number of washing plus centrifugation steps.   Keep in mind that, while non-adherent cells need as little as 1 hour in PI to achieve tight CVs of their G1 and G2/M populations, adherent cells frequently need at least 3 or 4 hours.  For this reason, storing the cells overnight in Vindelov's PI solution in the refrigerator is a useful (and stress-reducing) step, but don't store them more than 2 days, as many stained cells will then begin to show signs of falling apart by day 3 and thereby increase background noise in the sub-G1 region.     Good luck. 

Dear Salman1

Natural sedimentation velocity of cells (1g) of about 1 mm per minute.

You can easily check it if you directly observed isolated living cells in an inverted microscope. When the thickness of the layer of culture medium in a bottle or PBS is 5 mm  most of the cells will be after five minutes on the surface of the vial. In the medium volume will remain single cells. Therefore centrifugation 500 g per minute gather cells from the liquid column of about 500 mm. Therefore, I recommend not centrifuged  living cells with accelerations of more than 500 g. It will increase cell survival.

Best wishes

Rustem Uzbekov

You have to prepare cells for flow cytometry analysis quickly,  at low temperature, and centrifuge cells at 1000 rpm till 5-7 min

For definiteness, you must specify the parameters of centrifugation in "g".

Without data about type of  centrifuge and radius of the rotor the data in "rpm"  do not make sense. For example for our centrifuge Sigma 1-15PK 1000 RPM= 91 g  and for our centrifuge Heraeus Megafuge 16R : 1000 RPM = 184g.

Dear Salman

Neutralizing the trypsin with FCS instead of sedimentation, would save one centrifugation step and might decrease the rate of dead cells.

Dear Salman!

You can minimize the processing time with trypsin. Shakes vigorously cells after a short incubation instead of the long incubation in trypsin. Then add the serum immediately  (I agree with Amir). Centrifugation was carried out at the minimum acceleration sufficient for sedimentation of cells. Try 100 g or less.

Best wishes

Rustem Uzbekov