Eight peak rainbow beads

All cell sorting experiments were performed on a BD Influx cell sorter running BD FACS Sortware. Laser alignment was performed using eight-peak rainbow beads (Spherotech), and drop delay was determined using BD Accudrop beads.
The plate holder apparatus on a BD Influx does not hold a nonskirted 96-well PCR plate tightly. To create a fitting holder, a 96-well polycarbonate rack typically used to hold individual 1.4-mL polypropylene round-bottom tubes was used. By removing the legs of the rack and shaving the bottom surface to be flat, we were able to create a rigid fit in the sort tray of the Influx sorter. Standard 96-well PCR plates were able to fit easily into the rack and were secured using individual portions of a pressure-sensitive adhesive (e.g., Blu-Tack) in several locations within the rack. To establish the alignment of the sort plate on the sort stage we performed sorts of 10 beads onto the lid of a 96-well plate. Cells were then index sorted into wells of a 96-well plate and analyzed further. To determine the precision of the cell sorter, cells were index sorted into 96-well PCR plates to perform Fluidigm real-time PCR analysis (Fig. 1C).

Populations of Venus-positive cells (L cells) or Venus-negative cells (non-L cells) of purity greater than 90% were separated from the tissues of GLU-Venus mice using a BD Influx cell sorter running BD FACS Software as previously described (16 (link)). Laser alignment was performed using eight-peak rainbow beads (Spherotech), and drop delay was determined using BD Accudrop beads. RNA was extracted from FACS-sorted cells by a microscale RNA isolation kit (Ambion) and reverse transcribed to cDNA according to standard protocols. A first-strand cDNA template was mixed with specific TaqMan primers (Applied Biosystems), water, and PCR master mix (Applied Biosystems), and quantitative RT-PCR was conducted using a 7900HT Fast real-time PCR system (Applied Biosystems). β-Actin was used as the normalization control. The primer/probe pairs used in this study were from Applied Biosystems: Agtr1, Mm01957722_s1, and Mas1, Mm00434823_s1. All experiments were performed on at least three cDNAs isolated from one mouse each.