Ad libitum fed Pomc-eGFP mice were sacrificed by cervical dislocation, and tissue from the medial basal hypothalamus (MBH) region was micro-dissected into Hibernate A medium without calcium and magnesium (BrainBits, Springfield, IL, USA). The tissue was then digested with Papain (20 U/ml, Worthington, Lakewood, NJ, USA) for 30 min at 37 °C with mild agitation, followed by trituration in Hibernate A medium containing DNase I (0.005%, Worthington). The cell suspension was then filtered through a 40 μm cell strainer into a new Falcon tube.
Fluorescence-activating cell sorting was performed using an Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) utilizing a protocol that was previously used for single-cell isolation [31] (link), [32] (link). The cell gating was set according to cell size (FSC), cell granularity (SSC), FSC pulse-width for singlets, fluorescence at 488 nm/532 nm for GFP and 647/670 nm for nuclear stain with DraQ5 (Biostatus, Shepshed, Leicester, UK). Cells were sorted direct into a 96 well plate containing lysis buffer with RNase inhibitor.
Fluorescence-activating cell sorting was performed using an Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) utilizing a protocol that was previously used for single-cell isolation [31] (link), [32] (link). The cell gating was set according to cell size (FSC), cell granularity (SSC), FSC pulse-width for singlets, fluorescence at 488 nm/532 nm for GFP and 647/670 nm for nuclear stain with DraQ5 (Biostatus, Shepshed, Leicester, UK). Cells were sorted direct into a 96 well plate containing lysis buffer with RNase inhibitor.
Free full text: Click here
