Facsaria 1 sorp sorter

FACS was performed as in Castellanos et al. (2008) (link). Briefly, brain lobes or allograft-derived tumors were dissected in Rinaldini's solution (800 mg NaCl, 20 mg KCl, 5 mg NaH₂PO₄, 100 mg NaHCO₃, 100 mg glucose in 100 ml distilled H₂O) and incubated at 30°C for 60 min in dissociation solution made of complemented Schneider's medium (5 ml fetal bovine serum, 0.1 ml insulin, 1 ml PenStrep, 5 ml L-glutamine and 0.4 ml L-glutathione in 37.85 ml Schneider's medium) supplemented with 1 mg/ml papain and 1 mg/ml collagenase. The dissociation solution was then replaced with complemented Schneider's medium and tissues were disrupted by repeated pipetting. Flow cytometry was carried out using a FacsAria I SORP sorter (Beckton Dickinson) at the Cytometry Unit, Centres Científics i Tecnològics (CCiT) of the University of Barcelona. 488 nm and 561 nm excitation laser beams were used for forward and side scatter (FSC and SSC), respectively. Cells were gated according to their FSC versus SSC parameters; doublets were excluded using TOF parameter. Brain lobes from w1118 larvae and Tub-GAL4 UAS-CD8::GFP heterozygous larvae were used to gate GFP-negative and -positive populations, respectively. A 70 μm nozzle was used to sort GFP⁺ or GFP⁻ cells. FlowJo software was used to visualize the flow cytometry data (FlowJo 10.7.2, https://www.flowjo.com/citing-flowjo).

Wing imaginal discs of each genotype were dissected in cold PBS, dissociated with trypsin-EDTA at 32°C for 45 min, and fixed with 4% formaldehyde for 20 min. Cells were centrifuged at 2,000 rpm for 2 min, resuspended in 1 ml of 70% ethanol, and incubated for 1 h at room temperature (RT). After centrifugation, the pellet was resuspended in PBS with DAPI 1 mg/ml and RNase 100 mg/ml and incubated for 1 h at RT. DAPI fluorescence was determined by flow cytometry using a FacsAria I SORP sorter (Beckton Dickinson, San Jose, California). Excitation with the blue line of the laser (488 nm) permits the acquisition of scatter parameters. Blue laser (488 nm) was used for GFP excitation, and a UV laser (350 nm) for DAPI excitation. Doublets were discriminated using an integral/peak dot plot of DAPI fluorescence. Cell cycle histograms were obtained on each sample according to the GFP fluorescence, and cell cycle analysis was done on DAPI fluorescence collected at 440 nm. DNA analysis on single fluorescence histograms was done using Summit software (Dako Colorado).