Cook Book Sept 2010 Volume 12
For the majority of manipulations using cell cultures, such as transfections, cell fusion techniques, cryopreservation and subculture routines it is necessary to quantify the number of cells prior to use. Using a consistent number of cells will maintain optimum growth and also help to standardise procedures using cell cultures. This in turn gives results with better reproducibility.
- Personal protective equipment (sterile gloves, laboratory coat, safety visor)
- Waterbath set to appropriate temperature
- Microbiological safety cabinet at appropriate containment level
- CO2 incubator
- Inverted phase contrast microscope
- Pre-labelled fl asks
- Media– pre-warmed to appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and temperature)
- 70% (v/v) isopropanol in sterile water
- 0.4% Trypan Blue Solution
- Bring adherent and semi-adherent cells into suspension using trypsin/ EDTA as described previously (Protocol 3 and 4) and resuspend in a volume of fresh medium at least equivalent to the volume of trypsin. For cells that grow in clumps centrifuge and resuspend in a small volume and gently pipette to break up clumps.
- Under sterile conditions remove 100-200μl of cell suspension.
- Add an equal volume of Trypan Blue (dilution factor =2) and mix by gentle pipetting.
- Clean the haemocytometer.
- Moisten the coverslip with water or exhaled breath. Slide the coverslip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the coverslip).
- Fill both sides of the chamber with cell suspension (approximately 5-10μl) and view under an inverted phase contrast microscope using x20 magnifi cation.
- Count the number of viable (seen as bright cells) and non-viable cells (stained blue). Ideally >100 cells should be counted in order to increase the accuracy of the cell count (see notes below). Note the number of squares counted to obtain your count of >100.
- Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below.
|Viable Cell Count (live cells per millilitre) =||Number Live Cells Counted||x Dilution x 10,000|
|Number of large corner Squares counted|
|Non-viable Cell Count (dead cells per millilitre) =||Number Dead Cells Counted||x Dilution x 10,000|
|Number of large corner Squares counted|
|Percentage Viability =||No of Viable Cells||x 100|
|Total No of Cells|
A haemocytometer being filled with cell suspension
Dimensions of a haemocytometer
Grid on a haemocytometer
Guide to which cells to count on grid i.e. include cells in middle of grid and cells that overlap edge on two sides (ticked), exclude cells that overlap edge on other two sides (crossed)
- Trypan Blue is toxic and is a potential carcinogen. Protective clothing, gloves and face/eye protection should be worn. Do not breathe the vapour.
- The central area of the counting chamber is 1mm2. This area is subdivided into 25 smaller squares (1/25mm2). Each of these is surrounded by triple lines and is then further divided into 16 (1/400mm2). The depth of the chamber is 0.1mm.
- The correction factor of 104 converts 0.1mm3 to 1ml (0.1mm3 = 1mm2 x 0.1mm)
- There are several sources of inaccuracy:
- The presence of air bubbles and debris in the chamber.
- Overfilling the chamber such that sample runs into the channels or the other chamber
- Incomplete filling of the chamber.
- Cells not evenly distributed throughout the chamber.
- Too few cells to count. This can be overcome by centrifuging the cells, re-suspending in a smaller volume and recounting.
- Too many cells to count. This can be overcome by using a higher dilution factor in trypan blue e.g. 1:10
- The use of a haemocytometer can be time consuming, susceptible to subjective judgements by the operator and some cell types, such as those that form clusters, are particularly diffi cult to count using this method. Technology which counts cell nuclei, such as the NucleoCounter (Chemometec), is available offering an alternative cell quantifi cation method. Unlike other cell quantifi cation methods, automated nuclei counters eliminate manual counting and do not rely on the retention of physical and/or morphological properties of the cells. Low volumes, e.g. 200μl, of prepared samples are drawn into a cassette which is inserted into the nuclei counter providing a cell count in approximately thirty seconds.