For the majority of manipulations using cell lines, 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.
1. Trypan Blue 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 1 mm2. This area is subdivided into 25 smaller squares (1/25 mm2). Each of these is surrounded by triple lines and is then further divided into 16 (1/400 mm2). The depth of the chamber is 0.1 mm.
3. The correction factor of 104 converts 0.1 mm3 to 1 mL (0.1 mm3 = 1 mm2 x 0.1 mm)
4. There are several sources of inaccuracy:
5. The use of a hemocytometer can be time consuming, susceptible to subjective judgements by the operator and some cell types, such as those that form clusters, are particularly difficult to count using this method. Cell counting equipment is available offering alternative cell quantification methods including the Scepter™ Cell Counter. The Muse® Cell Analyzer enables flow cytometry-based assessment of cell count and viability. The Scepter™ Cell Counter is a portable, handheld cell counter that measures volume using the Coulter Principle. It can quantify cells based on size and will discriminate larger cells from smaller debris, unlike vision-based techniques, which rely on object recognition software and cannot reliably detect small cells. The Scepter™ cell counter detects every cell and displays the population as a histogram of cell size distributions. From the histogram, count all the cells or use the gating function to count a chosen subpopulation. By monitoring changes in your histogram, you can gain insight into the health and quality of your cell culture from one experiment to the next.
Figure 1.Counting cells using a hemocytometer and trypan blue. Viable cells contain intact cell membranes and do not uptake trypan blue, appearing bright/clear in the hemocytometer. Dead cells have damaged cell membranes and uptake trypan blue, appearing blue in the hemocytometer. Cell viability can be estimated by taking the ratio of live/dead cells.