2.1 Tissue sources and pancreatic cell isolation
Animal care and immunization procedures were performed in accordance with the institutional review committee at Oregon Health & Science University. BALB/cBy, 129/S3, and NOD.Cg-Prkdcscid Tg(Ins1-EGFP/GH1)14Hara/Sz (“MIP-GFP”) mice were obtained from the Jackson Laboratory. F344 rats were acquired from Charles River Laboratories. Adult pancreatic tissue was collected from c129/S3 mice aged 2–4 months. For the optimal preparation of a single cell suspension of whole pancreas tissue, a modified perfusion digest was first employed. This involved cannulation of the portal vein and the sequential administration of Ca2+/Mg2+-free EBSS (4’; Gibco) followed by a digest solution containing 0.1 mg/ml Collagenase XI (12’; Sigma-Aldrich) in regular EBSS at 2.5 ml/minute. The organ was then removed and manually dispersed using forceps and pipetting action (a p1000 minipipetter with a clipped disposable tip). Remaining solid tissue was subjected to in vitro dissociation (30’) with a solution of 2.5 mg/ml Collagenase D (plus 0.1 mg/ml Dnase I and trypsin/chymotrypsin inhibitor [all Sigma-Aldrich]). Fully dissociated cells were collected after perfusion (fraction 1) and after in vitro dissociation (fraction 2) by passage through a 40 µm cell strainer (BD Falcon) and stored without further enzyme exposure. For immunofluorescent screening, pancreata were collected from wild type or MIP-GFP mice (also aged 2–4 months), embedded in Tissue-tek cryomatrix (Sakura, Tokyo, Japan), and stored at −80°C.
2.2 Antibody production
A modified subtractive immunization protocol (Williams et al., 1992
) was employed. Specifically, a F344 rat was pre-immunized with undesirable antigens including partially dispersed whole-pancreatic tissue, fetal bovine serum (FBS), and small amounts of collagenase. Cyclophosphamide (Sigma-Aldrich; 51 mg/kg) was then injected IP after 24h and 48h to eliminate B lymphocytes reacting against these antigens. Immunization of purified mouse islets was performed intraperitoneally (IP) on day 19 (dose #1) and day 38 (boost) after the initial treatment. On day 42, the rats were sacrificed and their spleens were harvested. Splenocytes were fused with SP2/0 Ag14 myeloma cells and successfully fused clones were selected by growth in methylcellulose-containing HAT medium (Stem Cell Technologies Inc., Vancouver, Canada). Approximately 500 isolated clones were transferred to liquid media in 96w plates; supernatants were collected for screening by immunofluoresence (IF) on sections of mouse pancreas and by flow cytometry on dispersed viable mouse pancreatic cells. Clones of particular interest were cryopreserved and passaged to larger culture flasks for increased supernatant production.
2.3 Immunofluorescent imaging
Cryosections (5 µm) of mouse pancreas were prepared using a Reichert 2800 Frigocut cryostat (Reichert Scientific Instruments) and fixed with acetone (5’) at −20°C. After drying, slides were stored at −86°C for up to three months. Labeling was performed with 50 µl of 1:20 diluted hybridoma supernatant (30’). Slides were then washed in DPBS and labeled (20’) with 50 µl of secondary antibody solution (1:200 dilution of Cy3-conjugated goat anti-rat IgG [Jackson ImmunoResearch, West Grove PA]) and 2% FBS [Hyclone]). DPBS was used for a final wash and storage of the slides prior to evaluation with a Zeiss Axioskop 2 plus (Carl Zeiss, Jenna, Germany). For dual antibody labeling, rabbit anti-mouse CK19 or rabbit anti-mouse amylase was included in the primary antibody labeling and Alexa488-conjugated goat anti-rabbit IgG (Invitrogen) was added for the secondary antibody labeling. All sections were mounted in a solution containing 10% glycerol and 4% N-propyl gallate (Sigma-Aldrich) with 0.001% Hoechst 33342 as a nuclear label. Antibodies specific for mouse DGKB (Abgent Inc., San Diego, CA), GPM6a (MBL International Corp., Woburn, MA), DPPIV/CD26 (BD Biosciences) and TTR (Lifespan Biosciences, Seattle, WA) were used on mouse pancreatic cryosections fixed with 10% formalin and detected using the secondary antibodies described above.
2.4 Flow cytometry and FACS
Dissociated cells were resuspended at 1x106 cells/ml in DMEM + 2% FBS + 0.1 mg/ml trypsin/chymotrypsin inhibitor prior to the addition of hybridoma supernatant (at a 1:20 dilution) and storage at 4°C (for 30’). After a wash with cold DPBS, the cells were resuspended in DMEM + 2% FBS containing a 1:200 dilution of APC-conjugated goat anti-rat secondary antibody adsorbed against mouse serum proteins (Jackson Immunoresearch, cat# 712-136-153). After another wash, cells were resuspended in DMEM + 5% rat serum (Serotec) and held on ice (10’) to block the secondary antibody. A final incubation with Alexa488-conjugated anti-CD45 and Alexa488-anti-CD11b/Mac1 (Invitrogen) facilitated exclusion gating of hematopoietic cells. Propidium iodide staining was used to label dead cells for exclusion. Cells were analyzed and sorted with a Cytopeia inFluxV-GS (Becton-Dickenson, Franklin Lakes, NJ); FSC:PW gating was used to exclude cell doublets from analysis or collection.
2.5 RNA isolation and qRT-PCR
For molecular analysis, populations of interest were sorted directly into Trizol LS (Invitrogen). RNA was collected after chloroform extraction, glycogen-assisted isopropanol precipitation, and a 70% ethanol wash. First strand cDNA synthesis was generated by MMLV reverse transcriptase and random oligonucleotide primers (Invitrogen). Gene expression was quantified by qRT-PCR using a Bio-Rad iQ5 thermocycler with a single-color MyiQ detection system. All reactions were performed in 45 cycles (15” @ 95°C, 20” @ 68°C, 20” @ 72°C). Reaction mixtures included 1.5u Platinum Taq DNA polymerase (Invitrogen), 2.5 mM MgCl2, 10 µM 5’ and 3’ primers, 10 mM dNTPs and 0.5x SYBR green. All primers were designed and tested to specifically amplify cDNA products of RNA encoding Pancreatic amylase 2 (5’: TGGCGTCAAATCAGGAACATGG, 3’: GGCTGACAAAGCCCAGTCATCA), Glycoprotein 2 (5’: AGGAGCCGAAGTGTTGCTTCCA, 3’: TCACGTTGGTTTGGGCATCTGT), CK19 (5’: GGACCCTCCCGAGATTACAACCA, 3’: GCCAGCTCCTCCTTCAGGCTCT), Cystic Fibrosis Transmembrane Receptor (5’: TCTCAGCCTTCTGTGGCCTTGG, 3’: TCCGGGTCATTTTCAGCTCCAC), von Willenbrand factor (5’: TGTGGGCTGTGCGGTGATTTTA, 3’: TGGGAGGAGATGCCCGTTTACA), Insulin 1* (5’: AGACCTTGGCGTTGGAGGTGGCCCG, 3’: GCAGAGGGGTGGGGCGGGTCGAG), Insulin 2* (5’: CCTGCCCCTGCTGGCCCTGCTCT, 3’: CCCGGGCCTCCACCCAGCTCCA), Glucagon (5’: ACCTGGACTCCCGCCGTGCCCA, 3’: TCGCCTTCCTCGGCCTTTCACCAGCC), Somatostatin* (5’: TGGCTGCGCTCTGCATCGTCCTGGCT, 3’: TGACGGAGTCTGGGGTCCGAGGGCG), Pdx1* (5’: GCGGTGGGGGCGAAGAGCCGGA, 3’: GACGCCTGGGGGCACGGCACCT), TTR (5’: GCGGAGTCTGGAGAGCTGCACGGGCT, 3’: TGGGCTGAGCAGGGCTGCGATGGT), DPPIV (5’: GGCCCTGCGTGCTACTTCCTGGCTCG, 3’: ACGTCCTGCGCGGCTGCTCTGC), DGKB (5’: GCCCGCTCTTCTTTCAGGTGGT, 3’: GGTGGATCACTTCTGGGGAGCA), GPM6A (5’: GTGGCAGATGTGTGAGCGCTTG, 3’: TGTCACAATCCCAAACTGACGCA), and GAPDH* (5’: AAGGTCGGTGTGAACGGATTTGG, 3’: CGTTGAATTTGCCGTGAGTGGAG). Except where noted (asterisks), each amplicon spans a >2kb intron. Where possible, one primer lies on an intron-exon boundary to further minimize amplification of contaminating genomic DNA. Cycle threshold values were recorded as baseline corrected curve-fitted values and reported as normalized values relative to housekeeping gene GAPDH.