DNA synthesis in cultured neonatal rat islets--a comparison of two methods.
To assess the effect of glucose, foetal calf serum (FCS) and iron-saturated transferrin (Fe-TRF) on DNA synthesis of fibroblast-free neonatal rat islets cultured in Ham's F-12 medium, we measured [3H]thymidine uptake in cultures and used immunohistochemistry to quantitate nuclear 5-bromo-2'-deoxyuridine staining. Addition of glucose to a concentration of 26.1 mmol/l resulted in a significant increase from baseline in DNA synthesis in islet cells as measured by both methods (22,591 counts/min per microgram DNA +/- 3628 S.E.M., vs. 9631 counts/min per microgram DNA +/- 1912 S.E.M., P < 0.002 and 19.19% positive beta-cells +/- 2.72 S.E.M., vs. 11.98 positive beta-cells +/- 0.26 S.E.M., P = 0.05). At a glucose concentration of 16.1 mmol/l we could demonstrate an increase compared with baseline only in [3H]thymidine uptake (15,700 counts/min per microgram DNA +/- 3323 S.E.M., P < 0.05). Supplementation with 10% or 15% FCS also increased [3H]thymidine uptake compared with baseline (to 15,809 counts/min per micrograms DNA +/- 136 +/- S.E.M., P < 0.05 and 23,746 counts/min per microgram DNA +/- 3114 S.E.M., P < 0.01, respectively) but did not significantly effect the BrDU labelling index. Addition of Fe-TRF to islet cultures significantly increased [3H]thymidine uptake at a concentration of 45 micrograms/ml compared with baseline (23,149 counts/min per microgram DNA +/- 6387 S.E.M., P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Hulinsky
I
I
R. Williams Institute of Paediatric Endocrinology, R.A.H.C., Comperdown, N.S.W. Australia.
Hulinska
H
H
Silink
M
M
D000818:Animals; D000831:Animals, Newborn; D001973:Bromodeoxyuridine; D002478:Cells, Cultured; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects; D005260:Female; D005947:Glucose / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D007501:Iron / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D011865:Radioisotope Dilution Technique; D051381:Rats; D017207:Rats, Sprague-Dawley; D013936:Thymidine / Q000378:metabolism*; D014168:Transferrin / Q000494:pharmacology; D014316:Tritium
D003470:Culture Media; D014168:Transferrin; D014316:Tritium; D004247:DNA; D007501:Iron; D001973:Bromodeoxyuridine; D005947:Glucose; D013936:Thymidine