Human islets obtained from Prodo Labs were dispersed using Accutase (MT25058CI, Thermo Fisher Scientific) and infected with an adenovirus expressing the bright green fluorescent protein, ZsGreen (Clontech, Mountain View CA) under control of the rat insulin-1 promoter (RIP1) and a mini-CMV enhancer. The RIP1-miniCMV promoter included 177 bases of the hCMV IE-1 promoter ClaI-SpeI fragment ligated to 438 bases of the RIP1 promoter44 (link),45 (link). Following transduction with the Ad.RIP-ZsGreen adenovirus for two hours, RPMI1640 medium containing 10% FBS was added to terminate adenovirus infection. Cells were collected 96 h after infection and loaded onto an Aria II cell sorter (BD biosciences), the live ZsGreen+ cell (β-cells) FITC-positive population was collected. The β-cell fraction was confirmed to be >92% pure by immunolabeling of sorted cells with insulin, and independently, by qRT-PCR and by RNAseq44 (link),45 (link). This procedure was repeated several times using islets from n=3 donors and sorted β-cells were pelleted and frozen at −80°C until enough starting materials for proteomic analyses were obtained. The donors were non-diabetic with BMIs of 26.3, 28.8, and 27 and ages 48, 61 and 63, respectively. Please see Supplementary method for gating strategy.
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Isolation and Purification of Human Islet β-Cells
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Variable analysis
independent variables
- Adenovirus expressing the bright green fluorescent protein, ZsGreen under control of the rat insulin-1 promoter (RIP1) and a mini-CMV enhancer
- Percentage of live ZsGreen+ cells (β-cells) in the FITC-positive population
- Islet samples from 3 non-diabetic donors with BMIs of 26.3, 28.8, and 27, and ages 48, 61, and 63, respectively
- RPMI1640 medium containing 10% FBS used to terminate adenovirus infection
- Positive control: Immunolabeling of sorted cells with insulin to confirm >92% purity of β-cell fraction
- Positive control: qRT-PCR and RNAseq to independently confirm >92% purity of β-cell fraction
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