A microassay to quantitate collagen synthesis by cells in culture.
A method to quantitate collagen synthesis, total protein synthesis, and DNA in 24-well culture plates is presented. Collagen-producing cells such as human intestinal smooth muscle cells and dermal fibroblasts were pulse-labeled with [3H]proline. After incubation, the plates were heated to 90 degrees C to stop isotope incorporation and sonicated to lyse the cells and an aliquot was removed for DNA quantitation. Carrier protein was added, all protein was precipitated by trichloroacetic acid, and unbound isotope was removed by repeated precipitations. After incubation with purified bacterial collagenase, both the soluble 3H-labeled collagen-derived peptides and the remaining insoluble 3H-labeled noncollagen protein were quantified. Results were expressed as the amount of radioactivity incorporated into collagen and noncollagen protein per nanogram DNA and also as the percentage of collagen synthesis per total protein synthesized. The advantage of this technique over previous attempts to scale down the assay is that the entire assay for DNA, collagen, and non-collagen protein can be carried out in the same well without any transfer of material. This technique also provides a significant savings of culture medium, serum, growth factors, and cell material.
Diegelmann
R F
RF
Department of Surgery, Children's Medical Center, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0117.
Bryson
G R
GR
Flood
L C
LC
Graham
M F
MF
D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007422:Intestines / Q000166:cytology; D003012:Microbial Collagenase; D008832:Microchemistry; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D011392:Proline / Q000032:analysis; D014176:Protein Biosynthesis; D012867:Skin
D003094:Collagen; D004247:DNA; D011392:Proline; D003012:Microbial Collagenase