H-Thymidine Uptake by Cultured Cells

Materials

Fibroblast cells in log phase growth

Ca, Mg free-phosphate buffered saline (PBSA)

5% (w/v) Glutaraldehyde (GTA)

2% (w/v) Perchloric Acid (PCA)

Subbed slides (coated with chrom alum gelatin) and Permount

Nuclear Track Emulsion (Kodak or Ilford)

Darkroom and chemicals for photographic processing

Dektol developer

Kodak Fixer

Giemsa stain, graded series of alcohols, xylol

Procedure

1.Grow either L cells (Mouse fibroblasts) or chick embryo fibroblasts on coverglasses and then give them H-thymidine for a short period of time.

2.At the end of the labeling period, wash the coverslips in PBSA by gently grasping a coverslip with forceps and passing it through a beaker of saline.

3.Fix cultures in glutaraldehyde for 15 minutes.

4.Wash in several changes of water.

5.Wash in cold 2% PCA for 5 minutes to remove unincorporated labeled precursors to DNA. Repeat twice.