Influx high speed cell sorter

To perform a microarray analysis for genes expressed in circulating endothelial cells positive for CD109 (CD109+CECs) and CEC positive for CD146 (CD146+CECs), a three laser Influx high speed cell sorter (BD) was used.
Briefly, in two different experiments, 150 ml of blood from 8 healthy subjects were collected and after MNCs isolation with Ficoll- gradient, cells were depleted of white blood cells using anti-CD45 antibody coupled with magnetic MACS MicroBeads (Miltenyi Biotech) following the manufacturer's instructions. Cells were stained for Syto16, CD45, CD31 and CD109 and in the second experiment for Syto16, CD45, CD31 and CD146.
During sorting procedure, samples were continuously cooled to 4°C and a forward scatter pulse height and side scatter analyses were performed to exclude cell clusters and doublets. A two way cell sorting procedure was performed with a140 um nozzle with a 5.5 PSI pressure, and with an events rate of 1,000–1,500 events per second, using a sort pure mode. The sorting gate was painted on Syto16+CD45−CD31+CD109+ cells and on Syto16+CD45−CD31+CD146+ cells. Samples were collected into sterile polypropylene tubes containing RPMI and used for molecular analysis. Purity was always greater than 95% with a recovery of 70–80%.

E14 ESCs were cultured in DMEM containing 15% fetal calf serum and 10³ U/mL leukemia inhibitory factor (LIF); for 2i/LIF experiments, cells were cultured in serum-free N2B27 supplemented with LIF, 1 μM PD0325901, and 3 μM CHIR99021 inhibitors. The Zscan4c::emerald plasmid (90636) was kindly provided by Minoru Ko (Zalzman et al., 2010 (link)), and the MERVL::tdTomato reporter (Macfarlan et al., 2012 (link)) was a gift from Samuel Pfaff (Addgene #40281). Reporter stable clonal lines were generated by transfection (Fugene6, Promega), drug selection, and subcloning. Flow cytometry analysis was performed using the BD LSR Fortessa and sorts performed on the BD Aria III or BD Influx High-Speed Cell Sorter.

Flow cytometry was used to quantify the pigmented cells (i.e., phytoplankton) in the pyrosomes and seawater. Pyrosome flow cytometry samples were pulverized with a sterile pestle and filtered through a 50 µm filter to remove tissue that would clog the flow cytometer. A BD influx high-speed cell sorter (BD Biosciences, San Jose, CA) equipped with a small particle detector collected data triggered on forward-scattered light (FSC). For each particle, data on FSC, red fluorescence (chlorophyll), and orange fluorescence (phycoerythrin) were collected. Flow cytometry data were analyzed using FlowJo v10.6.2 and R v3.6.1 and deposited into FlowRepository #FR-FCM-Z3YE. Pigmented cells were defined based on their fluorescence signals above the autofluorescence of the macerated pyrosome tissue. Cells were classified as pigmented picoeukaryotes (PPE, chlorophyll only) or Synechococcus (chlorophyll and phycoerythrin). Welch’s t test was used to compare the relative abundance of Synechococcus to PPE in seawater vs. pyrosomes.

E14 mouse ESCs were grown under standard serum/LIF conditions (DMEM, 4,500 mg/L glucose, 4 mM L-glutamine, 110 mg/L sodium pyruvate, 15% fetal bovine serum, 1 U/mL penicillin, 1 mg/mL streptomycin, 0.1 mM nonessential amino acids, 50 mM b-mercaptoethanol, 10³ U/mL LIF). Single-MERVL::tdTomato and double-MERVL::tdTomato/Zscan4c::eGFP reporter cell lines were described in Eckersley-Maslin et al. (2016) (link), and Dux knockout cells were described in De Iaco et al. (2017) (link). Transfections were performed using Lipofectamine on preplated cells in six-well or 10-cm plate formats. Flow cytometry analysis was performed using BD LSR Fortessa, and sorts were performed on a BD Aria III or BD Influx high-speed cell sorter. siRNA transfections were performed by transfecting Dharmacon siRNA ON-TARGETplus siRNA SMARTpool at a final concentration of 50 nM with Lipofectamine.