Cell Quantification

 

Aim
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 standardize procedures using cell cultures. This in turn gives results with better reproducibility.

Materials

• Media� pre-warmed to appropriate temperature (refer to the ECACC Cell Line Data Sheet for the correct medium and temperature)
• 70% ethanol in water (Prod. No. R8382 )
• 0.4% Trypan Blue Solution (Prod. No. T8154 )
• Trypsin/EDTA (Prod. No. T4049 )

 

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)
• Waterbath set to appropriate temperature
• Microbiological safety cabinet at appropriate containment level
• Centrifuge
• CO₂ incubator
• Haemocytometer (Bright-line, Prod. No. Z359629 , Improved Neubauer, Camlab CCH.AC1)
• Inverted phase contrast microscope
• Pre-labeled flasks

 

Procedure

1. Bring adherent and semi adherent cells into suspension using trypsin/EDTA (Prod. No. T4049 ) as above (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.
2. Under sterile conditions remove 100-200uL of cell suspension.
3. Add an equal volume of Trypan Blue (Prod. No. T8154 ) (dilution factor =2) and mix by gentle pipetting.
4. Clean the haemocytometer.
5. Moisten the coverslip with water or exhaled breath. Slide the cover-slip 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 cover-slip).
6. Fill both sides of the chamber (approx. 5-10uL) with cell suspension and view under a light microscope using x20 magnification.
7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue) - (see below). 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.
8. Calculate the concentration of viable and non-viable cells and the percentage of viable cells using the equations below.

Where:

• A is the mean number of viable cells counted, i.e. Total viable cells counted divided by Number of squares
• B is the mean number of non-viable cell counted, i.e. Total non-viable cells counted divided by Number of squares
• C is the dilution factor and
• D is the correction factor (this is provided by the haemocytometer manufacturer).

 

Concentration of viable cells (cells/ml) = A x C x D
Concentration of non-viable cells (cells/ml) = B x C x D
Total number of viable cells = concentration of viable cells x volume
Total number of cells = number of viable + number of dead cells
Percentage Viability = (No of viable cells x 100) divided by Total No of cells

Key Points

1. Trypan Blue (Prod. No. T8154 ) is toxic and is a potential carcinogen. Protective clothing, gloves and face/eye protection should be worn. Do not breathe the vapor.
2. 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.
3. The correction factor of 10₄ converts 0.1mm₃ to 1ml (0.1mm₃ = 1mm₂ x 0.1mm)
4. 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, resuspending 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