Trypsinising/passaging adherent cell lines

Materials required:

Trypsin/EDTA

Sterile PBS

Cell culture flasks

10 and 25ml pipettes

5% Virkon

Trypan Blue/counting chamber (optional)

Growth medium (eg DMEM + 10%FCS/1%Pen+strep/1% glutamine)

Method

  1. Put trypsin, growth medium and PBS into 37C incubator for 45-60 minutes to warm before starting.
  2. Swab cabinet with 5% Virkon and 70% ethanol.
  3. Decant off spent media from each flask to be split.
  4. Add 25ml of warm PBS to each flask and wash monolayer gently. Decant off PBS into 5% Virkon.Add 5-10ml of trypsin to each flask. Lay the flask flat and wait for 1-2 minutes, or until most of the cells have rounded up (observe them under a microscope). Decant off excess trypsin into 5% Virkon, leaving about 1ml in the flask.
  5. Incubate flask at 37C for 1-2 minutes.
  6. Retrieve flask from incubator and knock the side of the flask against the palm of your hand a few times to dislodge the cells. The cells should come off easily - if not, reincubate the flask for a further 1-2 minutes.
  7. Resuspend the cells from each flask in 5-10ml of warm growth medium, and divide into labelled flasks. [If a cell count is to be performed, add 10m l of cell suspension to 90m l of Trypan Blue and mix well. Add 10m l to a haemocytometer and do a cell count. See below]
  8. Add 25-50ml of warm growth medium to the cell suspension in each flask, gently mix, and put flasks in 37C incubator with 5% CO2.

How to use a Haemocytometer

e.g. a Neubauer counting chamber

Count the number of non-blue cells in the shaded areas of the grid

 

 

 

 

 

 

 

 

 

 

Divide No.of cells by 5, multiply by dilution factor (10m l in 100m l = d.f of 10)

Multiply by 104. Result will be cells/ml.

e.g. 130 cells in 5 big squares, dilution factor of 10.

(130/5)x10x104=2.6x106 cells/ml