bind first Ab then radioactive second Ab stain and destain replica m expose replica to X-ray film expose replica to X-ray film compare film and replica for antigen-neg colonies

Fig. 2. Steps for detecting antigen in CHO cell colonies on polyester cloth replicas.

compare film and replica for antigen-neg colonies

Fig. 2. Steps for detecting antigen in CHO cell colonies on polyester cloth replicas.

Staining the Cells. The cells on the polyester disks are next stained for cellular protein with Coomassie blue G (Bio-Rad Laboratories) according to the technique of Raetz et al.1 The disks are stained with the dye (0.05 g/100 ml) dissolved in methanol/water/acetic acid, 45:45:10 (v/v) for 1 hr. The disks are then destained with several changes of the same solvent.

At this point the polyester disks radiolabeled for antigen and stained for total protein are dried on filter paper and attached to a sheet of paper. The outline of the disk including the notch and an identification number can be indicated with radioactive ink. The sheet with the polyester disks should be covered with plastic wrap before putting it against X-ray film. We use preflashed X-Omat AR film (Kodak) and expose at —70° with a Dupont Cronex screen. The length of time of exposure will depend on the amount of antigen present, the specific activity of the second antibody, as well as many other factors. The autoradiogram and the original polyester disks are then compared for colonies with altered antigen levels compared to Coo-massie blue staining. The colonies of interest can then be picked from the master dish using any one of a number of isolation procedures. If necessary the colony can be rescreened using this technique or by another assay until the desired colony is purified.

Work in Other Laboratories Using Replica Plating

A number of other laboratories have used the replica plating techniques described in this chapter. These groups used a variety of different cell lines and assay systems to isolate mutants. In this last section, a few of the different cell lines and assay strategies that have been used successfully will be described to give the reader a feel for the general versatility of the technique.

Chinese hamster ovary (CHO) cells appear to form the most reliable replicas and are very amenable to genetic manipulations. Another advantage of the CHO cells is the ability to make multiple high fidelity copies (up to 4) with the polyester. Other cell types that have been used in our laboratory include mouse fibroblasts (NIH 3T3 and Kirsten virus-transformed NIH 3T3), human HeLa (S3), and monkey (CV-1) cells. None of these cell lines transfers with as high efficiency as the CHO cells in this system. These cells make acceptable single copies with the 1 fim pore size material, but do not form reliable copies beyond that. Other laboratories have found similar problems with non-CHO cells. Raetz et al} described replica plating of a mouse myeloma line (SP210) and several hybridomas derived from the fusion of this line and mouse spleen cells. These workers supplement the growth medium with 2 mM CaCl2 and 10% BSA and make a single replica with the 1 fim polyester cloth. Dr. A. Garcia-Perez (Laboratory of Kidney and Electrolyte Metabolism, NIH) working with kidney cells (pig, LLCPK, and dog, MDCK) (personal communication) and Dr. F. Amano (National Cancer Institute, NIH) with murine macrophages (personal communication) both indicate that these cells make single replicas with the 1 /xm pore size polyester. Other workers have published work using replica plating of murine fibrosarcoma cells (HSDMiQ).7

Several laboratories have used replica plating to isolate a variety of

7 E. J. Neufeld, T. E. Bross, and P. W. Majerus, J. Biol. Chem. 259, 1986 (1984).

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