snap surface block  (New England Biolabs)

Journal: Molecular Metabolism

Article Title: Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

doi: 10.1016/j.molmet.2022.101541

Figure Lengend Snippet: Development of a MIN6 reporter cell line. (A) Schematic of the reporter construct (Created with BioRender.com ). (B) The workflow of establishing reporter cell clones. (C) Characterization of glucose induced insulin secretion in 20 clones. The luminescence intensity of NanoLuc was detected after 30min stimulation of 1 mM glucose (black, n = 4) or 25 mM glucose (gray, n = 4). (D) Immunoblot of Cas9 protein in control cells and clone #4, #6, #7, #8, #13, #14 and #15. (E) Representative confocal images of MIN6-6 after 20 min incubation with SNAP surface dye Alexa Fluor 546 together with 1 mM glucose or 25 mM glucose. Phogrin-EGFP was also imaged. Scale bar, 5 μm. (F) Flow cytometry analysis of MIN6-6 cells labeled with Alexa Fluor 546 in 1 mM glucose for 20 min, followed by labeling with Alexa Fluor 647 in 25 mM glucose (red), 25 mM glucose plus 300 μM Diazoxide (blue), or 40 mM KCl (orange).

Article Snippet: SNAP substrates were purchased from NEB (SNAP-Surface® Block, S9143S; SNAP-Surface® Alexa Fluor® 546, S9132S; SNAP-Surface® Alexa Fluor® 647, S9136S).

Techniques: Construct, Clone Assay, Western Blot, Incubation, Flow Cytometry, Labeling