Islets were isolated as described earlier . Informed consent was obtained from tissue donors and institutional Ethics Committees in Uppsala and Helsinki approved all procedures. Upon arrival, islets were cultured for 4 days in non-adherent culture dishes (Barloword Scientific, Stone, UK) in serum-free Ham’s F10 (10 mmol/l glucose; Sigma, St Louis, MO, USA) containing 0.5% bovine serum albumin (Sigma).
Initial cell clusters were dissociated with 0.16 mg/ml trypsin and 0.1 mmol/l EDTA and filtered through a 70 μm nylon mesh (Becton Dickinson Biosciences, Bedford, MA, USA).
Cell sorting and culture
For direct cell sorting, dispersed islets were incubated for 15 min with microbeads conjugated to PSA-NCAM (Miltenyi Biotech, Auburn, CA, USA). After a brief centrifugation at 300 g, cells were incubated for another 15 min with goat anti-mouse IgG microbeads (1:5; Miltenyi Biotech) in 250 μl of phosphate buffered saline. Cell separation was carried out on a MiniMACS magnetic cell separation system according to manufacturer’s instructions (Miltenyi Biotech).
For two-step cell sorting, dispersed islet cells were first incubated for 5 min at 4°C with mouse anti-human CA19-9 antibody and cell separation carried out as above. Negative cell fraction was immediately used for PSA-NCAM sorting as described above. All procedures of cell sorting except incubations were done at room temperature to minimise cell death .
Prior to immunostaining, purified cells were cultured for 4 days in 48-well dishes (Nunc, Roskilde, Denmark) in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% FCS (vol./vol.; PromoCell, Heidelberg, Germany).
Cells were fixed in 4% paraformaldehyde (wt/wt) and permeabilised with 0.1% Triton X-100 (vol./vol.). Pancreatic sections were deparaffinised and rehydrated before staining. Microwave treatment in citrate buffer was necessary to retrieve antigenicity of NCAM, CA19-9 and PSA-NCAM.
Non-specific binding was blocked by preincubation in 5% normal serum (vol./vol.; Zymed, San Francisco, CA, USA) of the species in which secondary antibody was raised. Specimens were incubated for 1 h with primary antibody followed by secondary antibody incubation for 45 min. The following primary antibodies were used: guinea pig anti-porcine insulin 1:200, rabbit anti-human glucagon 1:300, rabbit anti-human somatostatin 1:300, rabbit anti-human pancreatic polypeptide 1:300 (a mixture of glucagon, somatostatin and pancreatic polypeptide antibodies was used to simultaneously label alpha, delta and pancreatic polypeptide cells), rabbit anti-human chromogranin-A 1:500, mouse anti-human CK19 1:200 (all from Dako, Glostrup, Denmark); rabbit anti-α-amylase (1:300; Sigma); anti-PSA-NCAM (1:100; mouse monoclonal IgM; Chemicon, Billerica, MA, USA); anti-CA19-9 (1:100; mouse monoclonal; Novocastra, Newcastle upon Tyne, UK); and rabbit anti-NCAM 1:50 (kindly provided by V. Cirulli, The Whittier Institute for Diabetes, San Diego, CA, USA). Alexa fluor-conjugated goat anti-guinea pig, donkey anti-rabbit and donkey anti-mouse (1:300; all from Invitrogen), and FITC-conjugated rabbit anti-mouse IgM (Jackson Immuno Research Laboratories, West Grove, PA, USA) were used as secondary antibodies.