Abstract
Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, handpicked with a Pasteur pipette, and incubated (either intact or after dispersion with Dispase) for 0, 3, 5, 7, 10, or 14 days in tissue culture medium supplemented with either lymphokine supernatants or recombinant murine interferon-gamma. Islets and single cells were examined for IAk molecules by use of indirect immunofluorescence. Ia-positive islet cells were identified on the surface of islets incubated with 5-10% lymphokine for greater than 4 days or with 10, 100, or 1000 ng/ml interferon for greater than 6 days. Islets incubated in unsupplemented medium were Ia negative. Incubation with 5% lymphokine induced Ia expression on 10-40% dispersed islet cells cultured for greater than 9 days. Dual immunofluorescent staining for Ia and insulin revealed that Ia-positive cells included both beta- and non-beta-cells.
Highlights
We examined the effects of LK supernatants and recombinant IFN-7 on la antigen expression in isolated murine whole islets and dispersed islet cells
Dispase was inactivated with tissue culture medium
After 5 days of culture with 5% LK, many individual islet cells or groups of islet cells were outlined by irregular, granular fluorescent staining (Fig. 1)
Summary
METHODS AND MATERIALSIslets were isolated from male B10.BR/SgSnJ (H-2k) mice or Balb/c/J (H-2d) mice (Jackson Laboratories, Bar Harbor, ME) by the collagenase technique1718 and separated on a discontinuous ficoll gradient.[19]. AND ASSOCIATES gradient, washed, resuspended in tissue culture medium, and picked with a Pasteur pipette and dissecting microscope. Islets were dispersed into single cells immediately after isolation.[20] These islets were treated with room-temperature EDTA-versene (1:5000) for 7 min and digested with a Dispase (BoehringerMannheim, Indianapolis, IN) solution (10 mg/ml in calcium/magnesium-free medium) and gentle stirring. Single cells were collected by centrifugation, resuspended in medium, counted with a hemocytometer, and plated in plastic Petri dishes (2-5 x 105 cells/ml). Both intact islets and dispersed islet cells were incubated in CMRL-1066 medium containing 10% fetal calf serum, D-glucose (1.0 mg/ml), penicillin (100 U/ml), and streptomycin sulfate (100 |xg/ml)
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