Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes

Journal Article · Mon Feb 01 04:00:00 EST 1982 · Endocrinology; (United States)

DOI:https://doi.org/10.1210/endo-110-2-474· OSTI ID:5637963

Shimizu, F; Kahn, R

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH/sub 4/Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free (/sup 125/I)iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 x 10/sup 7//ml) consisted of incubation with (/sup 125/I)iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific (/sup 125/I)iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 x 10/sup 9//ml) were incubated with (/sup 125/)iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific (/sup 125/I)iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner.

• Research Organization: National Institutes of Health, Bethesda, MD
• OSTI ID: 5637963
• Journal Information: Endocrinology; (United States), Journal Name: Endocrinology; (United States) Vol. 110:2; ISSN ENDOA
• Country of Publication: United States
• Language: English

Related Subjects

551001* -- Physiological Systems-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
BETA DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY
BODY FLUIDS
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ERYTHROCYTES
HORMONES
INSULIN
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LEUKOCYTES
MAMMALS
MATERIALS
MICE
NUCLEI
ODD-EVEN NUCLEI
ORGANS
PEPTIDE HORMONES
RADIOISOTOPES
RADIORECEPTOR ASSAY
RODENTS
SPLEEN
TRACER TECHNIQUES
VERTEBRATES