Moflo astrios flow cytometer

For flow cytometry experiments, mammary glands were excised and digested as described for organoid experiments. Following digestion at 37 °C, samples were dissociated into single cells using TrypLE (Gibco, 12604-021), quenched with DMEM/10% FBS, and washed with PBS. Cells were then put through a 40 µM (Fisher, 22-363-547) or 70 µM strainer (Corning, 352350). For mammary gland profiling, live/dead staining was performed using a near-IR dead cell stain (Invitrogen, L34976), followed by blocking using Fc block (BD Biosciences, 553142) in BD stain buffer (BD, 554656). Samples were then incubated in primary antibodies at the specified concentrations for 30 min at 4 °C, washed twice, and resuspended in PBS. Compensation was performed using UltraComp eBeads Compensation Beads (Invitrogen). Flow cytometry was performed using a BD LSRII flow cytometer using FACS Diva 6.2.1 (BD Biosciences) and analysis was performed using FlowJo v10.7.1 (BD Biosciences). All antibodies used for flow cytometry are outlined in Supplementary Table 2. For FACS experiments, live/dead staining was performed using Calcein blue (Invitrogen, C1429) at a concentration of 1 µM. Cell sorting was performed on a Moflo Astrios flow cytometer (Beckman Coulter).

Sorting was performed using a MoFlo Astrios flow cytometer (Beckman Coulter) equipped with a 70 μm nozzle, operating at 35 psi. Standard gating procedures were used. Briefly, the first gate allowed separation of intact nuclei from debris. The second gate allowed us to identify individual nuclei, and exclude doublets and other aggregates. The third and fourth gates distinguish between PE+ and PE- nuclei and allowed us to sort and separate NeuN+ nuclei from NeuN- nuclei. Nuclei were sorted into 1 ml PBS (−Mg2+, −Ca2+) in a 2 ml polypropylene tube pre-coated with 200 ul of 5% BSA and rotated at 20 rpm at 4C.

For proliferation assays, CD27⁺ MPs were sorted with a MoFlo Astrios flow cytometer (Beckman Coulter, Brea, CA) equipped with a PMT-FSC detector, as previously described (13 (link), 15 (link)). Flow cytometer performance was assessed with Megamix-Plus FSC and SSC beads (BioCytex) before the MP sorting experiments. MPs were labeled with anti-CD27-BV510 (BD Biosciences) antibody (4°C, 30 minutes). We purified CD27⁺ MPs with diameters between 200 and 900 nm. We used a commercial kit to check for the absence of endotoxin in the sorted CD27⁺ MP preparations (In vivogen, San Diego, CA).

The use of human PBMC was approved by Institutional Review Board of the Catholic University of Korea (MC14TNSI0119) and written informed consent was obtained from participants. PBMCs were obtained from eight healthy donors, four of whom were male and four of whom were female. The median age was 31 years, with a range of 25 to 36 years. PBMCs were collected using Ficoll-Hypaque (GE Healthcare, Pittsburgh, PA, USA) and were preserved in liquid nitrogen. After thawing, PBMCs were stained with antibody and were sorted by flow cytometry (MoFlo Astrios flow cytometer, Beckman Coulter, Indianapolis, IN, USA). HLA typing was done by the Catholic Hematopoietic Stem Cell Bank (Seoul, Korea).