Cell plating efficiency

Chick embryo muscle cell plating efficiency was low on albumin and y globulin coated surfaces but the fibrinogen and gelatin coated surfaces were strongly cell adherent. 

Toxicity tests were conducted in Chinese Hamster Ovary cells to determine the concentration of either Ni3S2 or NiS which affected cell plating efficiency following a 24 h exposure period. Proliferating cultures of Chinese Hamster Ovary cells were exposed to the nickel compounds for 24 h, and then the cells were trypsinized from the monolayer. Cell numbers were determined with a hemocytometer and 400 cells were plated to form colonies in 100 irni diameter tissue culture plates. The cells were incubated for about 9 days, fixed, stained, and the total number of surviving colonies in each plate were counted and expressed as a function of the total number of cells plated. 

The literature is less extensive on the use of protoplasts in stress-tolerance investigations however, some applications have been attempted. For example, in one study protoplasts were isolated from the leaves of a wild relative of tomato shown to be salt tolerant and from a salt-sensitive, cultivated species (Rosen Tal, 1981). In the presence of NaCI the plating efficiency (number of surviving cells/number of cells applied to the plate) of the wild relative was greater than the cultivated, sensitive cultivar. Proline, when added to the culture media, was found to enhance the plating efficiency of the salt-sensitive cultivar but not the wild, salt-tolerant relative. These results suggest that traits related to salt tolerance are expressed by the isolated protoplasts and that the response of protoplasts to environmental stress can be manipulated, i.e. the proline response. 

The magnitude of the photodissociation effect is quite small in this case. A new IT ICR analyzer cell has recently been constructed that will permit passage of the laser beam through the cell without striking any of the cell plates. Gentle focussing of the pulsed laser beam should then increase the laser fluence in the center of the cell, and improve photodissociation efficiency. 

Prolonged and unnecessary enzymatic treatment, i.e., trypsinization of tumor cells, can also alter their survival and metastatic behavior in vivo. Moreover, viability tests (trypan blue exclusion) and even plating efficiency in vitro do not predict or correlate with the in vivo biological behavior of trypsinized cells. 

Plating efficiency experiments were performed by plating 200 viable cells into 60 mm tissue culture dishes and counting colonies after 7-10 days. Growth experiments were performed using microtiter wells with 1 x 10H cells/well on day 0 (N=3). 

Theoretically, the only conclusive signal of cell survival after drug exposure is the demonstration of reproductive integrity, which can be evaluated through plating efficiency and cell proliferation tests. Nevertheless, metabolic parameters could also be employed as a survival measure when the cell population is submitted to a recovery period after drug exposure. 

The number of cells per microcarrier bead, the concentration of beads and the initial stirring conditions can vary dramatically. The following is a suggested protocol but if plating efficiency is poor the culture volume and speed of stirring can be decreased and the number of cells per microcarrier increased. 

For measuring plating efficiency a similar procedure is adopted and the colonies are fixed and stained (with Giemsa) and their number compared with the initial input. The plating efficiency of primary cells is low, but plating efficiencies of up to 100% can be achieved with cell lines or cell strains. 

Release cells with 0.25% trypsin in PBS or BSS (the divalent ions improve plating efficiency) and disperse by gently pipetting up and down the minimum number of times. Trypsinisation at 4°C for 2-10 min has been recommended especially for cloning cells in low serum. 

Cryopreserved human hepatocytes Cryopreserved human hepatocytes are extremely useful for the evaluation of drug metabolism, but in general cannot be cultured due to their impaired cell-attachment. There are preparations of cryopreserved human hepatocytes with high plating efficiency and therefore can be cultured for induction studies. It has been shown that hepatocytes cultured after cryopreserva-tion are responsive to CYP1A and 3A inducers, but they have a significantly lower basal (uninduced) levels of these enzymes. Cryopreserved human hepatocytes therefore represent a more convenient experimental model than freshly isolated human hepatocytes for enzyme induction studies (Roymans etal. 2005). 

Harvest the cells and, if trypsin is used, add STI at 1 mg mH to prevent residual trypsin and STI from decreasing plating efficiency and viability. It is very important to neutralize the effects of trypsin and STI. This is done most effectively by centrifuging the cells after addition of STI to remove any residual trypsin and STI activity in the wash (McKeehan, 1977). 

Stir intermittently (for 1 min every 20 min) for the first 4-8 h. However, only use this option for cells with very poor plating efficiency because it causes clumping and uneven distribution of cells per bead... 

Attachment efficiency The percentage of cells plated (seeded, inoculated) that attach to the surface of the culture vessel within a specified period of time. The conditions under which such a determination is made should always be stated. 

To ensure that the results of an assay are valid, specific criteria have been determined. The mutation frequency of the positive control group should be twice that of the solvent control group. The solvent controls should have a spontaneous mutation frequency between 20 and 100 per 10 surviving cells. In addition, the plating efficiency of the solvent controls must be >0%. 

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