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Plating efficiency will determine how well single cells can survive and form colonies. Since the ratio of the volume of medium to the volume of the cells is very large, there is a minimal impact of the cells on their environment. Thus, there is little opportunity for the cells to metabolize and convert amino acids, to bind toxic components, or to secrete autocrine growth factors they may need for growth. Plating efficiency is thus a more sensitive measure of the cell's response to its surroundings. This may be the method of choice for determining the nutritional requirements of cells or for testing and comparing serum lots or for toxicity testing of compounds. There are many people who prefer this method for assaying the effects of growth factors. However, the optimal concentrations of nutrients and growth factors derived using plating efficiency may be inadequate to support cell growth to high densities. And for some cells the best-known growth conditions are still inadequate to support their survival at low cell density. This indicates that the medium lacks an essential component(s) that the cells are providing for themselves in an autocrine fashion. Fig-
The effect of conditioned medium on the plating efficiency of M2R melanoma cells. The two plates were plated at the same density in hormone-supplemented, serum-free medium.
The plate at right had 50% of the same medium conditioned for 24 hr by cells at high density.
One active factor in the conditioned medium is TGF-a made by the melanoma cells.
ure 5.4 shows the plating efficiency of melanoma cells with and without conditioned medium from high-density cultures of the same cell type.
Plating efficiency is expressed as a percentage:
colonies formed No, of cells plated
x 100 = Plating efficiency
1. Cell cultures
2. Six 100-mm tissue culture plates
3. 15-ml conical centrifuge tubes
4. 60 ml growth medium
5. Trypsin solution
6. Soybean trypsin inhibitor (if serum free)
7. Hemacytometer or electronic particle counter Reagents for Fixing and Staining
1. 10% Formalin
2. Crystal violet (0.1% in water)
1. Trypsinize as described previously. Check visually to make sure the cells are in a single-cell suspension.
2. Neutralize by resuspending in 10 ml of growth medium with serum in a conical 15 ml tube, or add 1 ml STI and resuspend in 10 ml of serum-free growth medium.
3. Wash by centrifugation at 900 rpm for 3"C4 min. Resuspend in 5"C10 ml of growth medium.
4. Count an aliquot of cell suspension.
5. Determine the volume needed to aliquot 100, 500, and 1000 cells to duplicate 100-mm plates.
Because these volumes can be necessarily small, it is convenient to dilute to the appropriate cell number in 1 ml, and add this volume to 9 ml of growth medium.
6. Resuspend the cells thoroughly in the 10 ml of medium by repeated pipetting to make sure the cells are dispersed. Plate the cells in an appropriate growth medium in the 100-mm plate.
7. Incubate the plates until the colonies are large enough to be visible to the naked eye (0.2"C1 mm in diameter) but not running into each other. The time required will depend on the cells but is usually around 10 days.
8. Wash the plates 1 x with PBS; remove the PBS and add enough 10% formalin to cover the plate. Fix for 10 min.
9. Remove the formalin and add enough crystal violet solution (2"C3 ml) to cover the plate. Leave 10 min, remove the crystal violet, and rinse under running water until no color is left.
10. Count the colonies with a marker pen or use a colony counter if available.
Conditioned medium can be used to grow the same cells at low density that may ordinarily require a high density to grow (for example, see Fig. 5.4). One also may use conditioned medium from one cell type to grow another type of cell in continuous or primary culture. In theory, one is allowing the conditioning cell culture to provide the growth factors or metabolic products needed by the culture grown in the conditioned medium. Conditioned medium is prepared using fresh medium in the following manner:
1. Allow a plate of the cells to be used to condition the medium to grow to near (at 70"C90%) confluency.
2. Wash the plate once with medium. Then, fresh growth medium (jA serum or hormone supplementation) is placed on the cells for 24"C48 hr. If the cells are very dense or metabolize the medium rapidly, then use the shorter time period for conditioning. The conditioned medium should not be very yellow (i.e., have a pH < 6.9) when removed from the cells. If the pH is too low, it indicates that the medium is exhausted and may therefore either contain a lot of debris and released proteases from dying cells in the original culture and/or not contain sufficient nutrients to support the growth of fresh cells.
3. This medium is collected, filtered through a 0.22-mm low-protein-binding filter to remove any floating cells and used for "conditioned medium" where described.