For all experiments a triple reporter mouse strain ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase (ProGcg-Venus/RIP-Cherry/Per2:Luc) was derived by crossing the ProGlucagon-Venus(ProGcg-Venus) reporter mouse (23) with Rat Insulin2 promoter (RIP)-Cherry (RIP-Cherry) (24) and Period2:Luciferase (Per2:Luc) mice (25). ProGcg-Venus and RIP-Cherry reporters exhibit a high specificity for α- and β-cells, respectively, while the fusion protein PER2:Luciferase, encoded by Per2:Luc, is a circadian reporter functionally indistinguishable from the wild-type PER2 protein. The overview of the experimental procedures is illustrated in Figure 1. All experiments were conducted on male mice aged 7–16 weeks under standard animal housing conditions comprising ad libitum access to food and water and 12 h light/12 h dark cycles. Islet isolations were performed during morning hours (07:00 a.m.–12:00 a.m.). To study circadian rhythms in freshly isolated α- and β-cells in vivo, mice were subjected to night-restricted feeding 2 weeks prior to the experiment and during the entire period of sample collection as described previously (26), with half of the animals entrained with inversed light-dark and feeding cycles during the same period.

Islets of Langerhans were isolated by a standard procedure based on collagenase (Type XI, Sigma) digestion of pancreas followed by Ficoll purification (27). Briefly, 2 ml of a 2-mg/ml collagenase solution [diluted in Hanks Balanced Salt Solution (HBSS) (Gibco), 4.2 nM sodium bicarbonate, 1 M CaCl₂, and 1 M HEPES (Gibco)] was injected retrogradely through the ampulla of Vater into the exocrine part of the pancreas, which was dissected and digested in a water bath at 37°C (11–14 min, exact time needs to be calibrated for every new batch of enzyme). The digested mixture was then washed in HBSS, supplemented with 0.3% free fatty acid bovine serum albumin (BSA) (Sigma) with subsequent Ficoll gradient centrifugation (densities: 1.108, 1.096, and 1.069). Islets, collected from the gradient, were then washed in HBSS supplemented with 0.3% BSA. Islet cells were gently dissociated by trypsin (Gibco), re-suspended in KRB solution (pH 7.4, BSA, 1.4 mM glucose and 0.5 mM EDTA), and filtered through a 70-μm cell strainer (Falcon). α- and β-cell populations were separated by flow cytometry fluorescence-activated cell sorting [FACS; Astrios Sorter (Beckman Coulter)], based on fluorescence intensity and wavelength, single cell nature, size, and viability. Purity of the sorted cells was further assessed by a second, additional FACS analysis. The viability of treated cells was evaluated by FACS with DRAQ7™ (Abcam) or DAPI (ThermoFisher) dyes.

Sorted cells were plated on 35 mm dishes and fixed in 4% paraformaldehyde during 30 min. For immunohistochemistry, the fixed cells were incubated with mouse anti-Glucagon (1:500, Sigma) or guinea-pig anti-Insulin (1:500, ThermoFisher Scientific) primary antibodies. The signal was revealed by Alexa-Fluor anti-mouse 568 or Alexa-Fluor anti Guinea-pig 488 secondary antibodies, respectively (1:1,000, Molecular Probes). Bright-field and fluorescent images were obtained with EvosTL fluorescent microscope (ThermoFisher) using 4×, 10×, or 40× objectives.

For the in vitro culture, intact islets or sorted cells were recovered in RPMI 1640 complete medium (11.2 mM glucose, 110 μg/ml sodium pyruvate) supplemented with 10% fetal calf serum, 110 U/mL penicillin, 110 μg/ml streptomycin, and 50 μg/ml gentamycin and attached to 35 mm dishes or multi-well plates (LifeSystemDesign) pre-coated with a laminin-5-rich extracellular matrix (28). For hormone secretion assays, approximately 15,000 cells were plated per dish, in three separated drops of 50 μl each. For bioluminescence recordings either 250 islets or approximately 50,000 separated cells were plated per well.

Total RNA was prepared from homogenized islet cells using RNeasy^® Plus Micro Kit (Qiagen). Ten nanograms of total RNA were reverse transcribed (PrimeScript RT reagent kit; Takara) and pre-amplified (TaqMan PreAmp Master Mix; Applied Biosystems) following the manufacturer’s instructions. Specific target gene mRNA levels were analyzed by real-time quantitative PCR using the LightCycler technology (LC480; Roche Diagnostics). Mean values of gene expression were calculated from technical duplicates of each qRT-PCR analysis and normalized to the housekeeping gene Hypoxanthine guanine phosphoribosyl transferase (Hprt) exhibiting no significant variability of its expression level throughout each experiment, and therefore served as internal control. Primers used for this study are listed in Table 1.

Insulin and glucagon basal secretion assays were performed on approximately 15,000 attached separated α- or β-cells at 3 days in vitro. For basal insulin secretion assessment, cells were washed in KRB solution (Krebs-Ringer bicarbonate, pH 7.4, supplemented with 0.3% BSA) containing 2.8 mM glucose for 1 h, with subsequent incubation in KRB solution, containing 2.8 mM glucose for 30 min at 37°C in a cell culture incubator. For glucagon assessment, cells were washed in KRB solution containing 7 mM glucose for 1 h, followed by additional 30 min incubation in the same solution at 37°C in a cell culture incubator. To assess the islet cell acute secretory response, FACS-separated α- (Venus-positive) and β- (Cherry-positive) cells were subjected to 2 h incubation in KRB solution containing 5.6 mM glucose, followed by additional 30 min incubation in KRB containing 5.6 mM glucose (basal condition), 30 min incubation in KRB containing 5.6 mM glucose supplemented with 10 mM arginine (stimulated secretion for Venus-positive cells) or in KRB containing 16.7 mM glucose (stimulated secretion for Cherry-positive cells), and additional 30 min incubation in KRB containing 5.6 mM glucose (re-basal condition). At the end of each experiment, cells were lysed in Acid/Ethanol mixture (1.5% HCl/75% Ethanol) for glucagon or insulin residual contents measurements. Insulin and glucagon concentrations were quantified in the supernatants and in lysed cells by the Mouse Insulin or Glucagon ELISA kits (Mercodia). For acute secretion assays, glucagon and insulin values were expressed as a percentage of appropriate released hormone to residual cell content.

Adherent islets/islet cells were synchronized by a 1-h pulse of forskolin 10 μM (Sigma) prior to continuous bioluminescence recording in RPMI, supplemented with 100 μM luciferin (NanoLight Technology) (20). Photon counts of each well were integrated during 1 min, over 24 min intervals. For detrended time series, raw luminescence signals were smoothened by a moving average with a window of 24 h, allowing for a less biased comparison of bioluminescence values across experiments with regard to the measured circadian parameters (20).

In order to simultaneously label α- and β-cells within the islets, a ProGcg-Venus/RIP-Cherry/Per2:Luc reporter mouse line was established (Figure 1), allowing for the highly specific separation of nearly pure endocrine cell populations. To this end, following islet isolation and gentle trypsinization, dispersed cells were sorted by FACS based on cellular fluorescence characteristics, cell size (based on the data of Forward Scatter detector, FSC), and granularity (based on the data of Side Scatter detector, SSC; see Figures 2A,B). Of note, Cherry-positive cells showed higher cell granularity than Venus-positive cells (compare two histograms in Figure 2B). In parallel, cell viability was assessed by utilizing DRAQ7™, a dye that binds to DNA when cell membrane permeability is altered after initiation of cell death. Overall viability across preparations was near 90% (Figure 2C), with almost a 10-fold higher percentage of cell death for Cherry-positive cells (up to 20%) than for Venus-positive cells (Figures 2D,E), suggesting a higher sensitivity of Cherry-positive cells to the islet isolation, trypsinization, and/or sorting processes. The average number of harvested viable Cherry-positive cells per mouse was 39,292 ± 6,887, which is more than threefold higher than for Venus-positive cells (12,521 ± 1,885) (Figure 2F), reflecting the physiological ratio between these two cell types within mouse islets (22). In addition, the purity of the obtained Venus- and Cherry-positive cell populations was assessed by a complementary round of FACS analysis (Figures 3A–G). According to this analysis, the α-cell population comprises more than 90% viable Venus-positive cells without detectable contamination with Cherry-positive cells (Figures 3A–C,G), while the β-cell population contained up to 96% viable Cherry-positive cells without visible Venus-positive contaminants (Figures 3D,E,G). Finally, the morphological examination of sorted cell populations with a fluorescent microscope confirmed their high purity (Figure 3H).

Insulin and glucagon transcript expression levels were assessed in separated Venus- and Cherry-positive cells by qRT-PCR analysis. As expected, Gcg transcription was the highest in the Venus-positive population, further confirming the α-cell identity, while both insulin transcripts Ins1 and Ins2 were abundant in Cherry-positive cells, indicating their β-cell identity (Figure 4A). Importantly, expression levels of the opposite cell hormone genes (Ins1 and Ins2 in Venus-positive cells, and Gcg in Cherry-positive cells) were more than 10-fold (for Ins1 and Ins2) and 1,000-fold (for Gcg) lower, further confirming the satisfactory purity of the α- and β-cell populations. Furthermore, Cherry-positive cell population expressed β-cell-specific transcription factor MafA at the levels which were 1,000-fold higher compared to this transcript expression in Venus-positive counterparts (Figure 4B).

For cell culture, separated α- and β-cells were plated on plastic dishes covered with laminin-enriched matrix, which improves islet cell survival and function, and keeping them from de-diffentiation (28). Attached islet cells were maintained in vitro for at least 10 days. During the first 48 h of culture, attached β-cells formed monolayer cell aggregates resembling pseudo islets, while α- cells formed small domed structures composed by a few cells or stayed separated (Figure 4C). Immunofluorescence analysis demonstrated that Venus-positive cells in culture co-localized with glucagon-specific antibody, whereas Cherry-positive cells co-localized with insulin-specific antibody (Figure 4D), further validating these cell identity and high purity of α- and β-cell fractions.

Hormone secretion assays, performed after 3 days in culture, detected high basal levels of insulin in the supernatant of β-cells, and high basal levels of glucagon in α-cell supernatants, released during 30 min in the presence of constant glucose concentrations (2.8 mM for insulin and 7 mM for glucagon; Figure 4E). Noteworthy, secretion of the opposite hormones (glucagon by β-cells and insulin by α-cells) was below the detection level. Importantly, incubation of cultured α-cells with arginine induced about 2.5-fold increase in glucagon secretion, whereas incubation of cultured β-cells with high glucose stimulated secretion of insulin above two-fold (Figure 4F). Taken together, these data suggest that the here-described methodology ensures highly specific and efficient separation of the two main populations of islet cells, resulting in nearly pure populations of viable and functional α- and β-cells, which can be maintained in culture.

In view of the complexity of the separation procedure by FACS, we next explored if separated α- and β-cell populations maintain their circadian properties, as was previously reported to be the case in isolated intact islets (11, 18). To this end, mRNA levels for selected core clock transcripts have been assessed in sorted islet cells isolated every 4 h during 24 h. The qRT-PCR analysis revealed pronounced rhythmic patterns for Per1 and Per2 genes over 24 h, while the oscillation of Clock transcription was shallow, in agreement with previous reports (Figure 5) (11, 19).

Moreover, we explored cell-autonomous molecular clocks in separated α- and β-cell primary cultures synchronized in vitro. Similar to intact islets (Figure 6A), populations of pure α-cells (Figure 6B) and β-cells (Figure 6C) responded to a 1 h synchronizing pulse of forskolin by demonstrating high-amplitude self-sustained circadian oscillations of Per2:Luc reporter expression for at least 5 consecutive days following synchronization. Synchronizing effect of forskolin on cultured α- and β-cells was specific, since medium change alone had little synchronizing effect on the Per2:Luc expression in both cell types (Figures 6B,C). Collectively, these data suggest that circadian oscillations persist in pure populations of α- and β-cells following islet isolation, trypsinization, and FACS separation procedures.