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While growth curves provide much information, they are too time and labor consuming to be used where changes in cell number are to be used as a screen. This may be the case if one wishes to assay the different fractions generated during the biochemical purification of a growth factor. Optimizing the nutritional components of a growth medium or screening conditioned media for growth-promoting activities also may be best performed using a
A typical growth curve of attachment-dependent cells. Note the lag phase before the cells start to grow, the log phase of growth, and the beginning of the plateau phase when contact-inhibited cells will cease growing. The population doubling time should be calculated during log phase growth and is 20 hr for this cell line (RL-65).
secondary endpoint for growth. Several of these will be discussed below in this section and more extensively in the section on high-throughput assays in Chapter 11. In addition to the changes in growth curves mentioned above, cells in different growth conditions can become multinucleate, increase or decrease total cell protein and volume, or undergo large changesin the levels of some mRNAs or enzymes. The secondary endpoint thus can vary to a different extent than the change in cell number or even in a different direction. Thus, if a secondary endpoint is to be used as a measure of cell proliferation, the assay used should be correlated with changes in cell number in both the control and test conditions.
Using the Hemacytometer or Electronic Particle Counter
Use of a hemacytometer or an electronic particle counter gives the most direct measurement of cell number and therefore cell growth. Cells can be counted before, during, and after setting up an experiment to accurately and directly quantitate and standardize experimental conditions. Moreover, the use of a dye such as trypan blue when doing hemacytometer counts gives the investigator a quantitative standard for the viability of the cells by doing a differential count of the cells that exclude trypan blue ("sort-of-sick" to viable) and those that take up the dye (irretrievably dead) (see also section on acridine orange'Cethidium bromide assay). The hemacytometer is undoubtedly the cheapest and most labor intensive method for counting cells, but it can be used to provide data as accurate as that obtained by any other method and to provide an assessment of both total and viable cell counts. This makes it an ideal method for the student laboratory or for laboratories where cell counts are not performed frequently.
1. Trypsinized cell cultures
2. Improved Neubauer hemacytometer with coverslip
3. Tally counter
4. 0.4% trypan blue in PBS
5. Pasteur pipettes
6. Microscope Procedure
1. Make a 1:1 dilution of cell suspension with 0.4% trypan blue. This can be further diluted with PBS if necessary.
2. Carefully resuspend with a Pasteur pipette.
3. Cover the hemacytometer chamber with the coverslip and place a drop of the suspension from the Pasteur pipette at the edge of the "V" shape on the chamber. Repeat for the other side of the chamber. It is important not to overfill or underfill, but rather to allow the drop to be drawn over the surface by capillary action.
4. Place the chamber on the stage of the microscope.
5. Initially focus on the etched lines of the chamber with low (4x) power (Fig. 5.2).
6. The hemacytometer consists of nine 1-mm squares that are divided into 25 smaller squares. The volume of one 1-mm square is 0.1 mm3 or 10'C4 ml. Using a 10x objective, focus on one of the 25 smaller squares bounded on all sides by three parallel lines.
The hemacytometer shown can be used as an inexpensive method to determine viable cell number.
For a viable cell number, count all the cells that exclude the dye. To total cell number, viable cell number, and percent viability, separately count the blue cells and those that exclude the dye. A multichannel push-button counter is very useful here. In order not to count cells that lie on the border (the three parallel lines more than once), make a point of counting those cells that lie on the top and the right and not on the bottom and left (this really depends on your orientation: Do it any way that is easy for you, just do not count the same cell twice).
7. Count at least 100 cells/mm2. If you count fewer than 100 cells in the square, count one or more additional squares.
8. Repeat for the second side of the chamber.
9. Calculate the number of cells/ml by multiplying the number of cells counted in 1-mm square (or the average of however many squares you counted) by 104.
Electronic Particle Counting
The Coulter counter, manufactured by Coulter Electronics, affords the investigator a rapid, accurate, and reproducible method of total cell counting, particularly when dealing routinely with a large number of samples to count. Several models are currently available that enjoy widespread popularity among cell culturists (Fig. 5.3). As a cell passes through the aperture, through which an electrical current is flowing, it displaces an equal volume of electrolyte. This causes a change of resistance in the path of the current and subsequently a change in the voltage. This change in voltage is directly proportional to the volume or size of the cell. Every change in voltage during the sample flow is represented as a cell