Moflo astrios eq flow cytometer

A Peyer’s patch sample from 2 days after infection, was homogenized in 1 ml of PBS for 2 × 55 s on a gentleMACS Octo Dissociator (Miltenyi Biotec) using a C-Tube (Miltenyi Biotec), which allows dissociation of viable single cells from tissue samples. Single cells were isolated by filtering through a 30-μm nylon mesh. Here, an aliquot of the filtered sample was diluted 200 times, and live/dead staining was performed. For this purpose, samples were incubated for 10 min with SYBR green I and PI to final concentrations of 1 and 2 μM, respectively.
The sorting was performed with a MoFlo Astrios EQ flow cytometer (Beckman Coulter) using the 488-nm laser and 530/40-nm filter (SYBR green I; all cells), 532-nm laser and 622/22-nm filter (PI; dead cells) for excitation and emission, plus a 70-μm nozzle, a sheath pressure of 60 lb/in², and 0.1-μm-filtered 1× PBS as sheath fluid. Forward scatter was used as the trigger channel. A tube sorting of single cells was performed with the most stringent settings (purify mode and 1- to 2-drop envelope) in order to obtain both a qualitative and quantitative measurement of the sorted sample. Cell sorting was performed at the Microbial Single Cell Genomics Facility at Science for Life Laboratory in Uppsala.

Mammary epithelial cells (MECs) and the separation of basal and luminal cells were performed essentially as described elsewhere^{3 (link), 54 (link)}. Mechanically dissociated inguinal mammary glands were digested on a shaking platform for 90 min at 37 °C in CO₂-independent medium (Invitrogen) containing 5% FBS, 3 mg/ml collagenase A and 100 U/ml hyaluronidase. Cells were pelleted, washed and resuspended in 0.25% trypsin-EDTA (Invitrogen) for 1 min, followed by incubation in 5 mg/ml dispase (Roche Diagnostics) containing 0.1 mg/ml DNase I (Sigma-Aldrich) for 5 min. Contaminating red blood cells were lysed using 0.17 mM NH₄Cl. Cells were stained using the following antibodies and dilutions: CD24-PerCP-Cy5.5 (1:200), CD49f-PE-Cy7 (1:50), CD45-APC (1:200) and CD31-APC (1:200) (Supplementary Table 1). DAPI was used to exclude nonviable cells. Basal (CD24^low/CD49f^high) and luminal (CD24^high/CD49f^low) cells were purified using MoFlo Astrios EQ Flow Cytometer (Beckman Coulter). Data were analysed with FlowJo software.

To overexpress circ-ATXN2, we used the pLC5-ciR-GFP (#GS0108) vector (Geneseed, Guangzhou, China). We inserted the full-length circ-ATXN2 (chr12: 40329454-40335680) into the pLC5-ciR-GFP vector between the restriction sites. Thereafter, the vector was packaged into the lentivirus and infected into rat ASCs (Sun et al., 2020 (link)). The vector-GFP- and circ-ATXN2–GFP-expressing cells were sorted for subsequent experiments using a Moflo Astrios^EQ flow cytometer (Beckman Coulter, United States).

Flow cytometry analysis was performed as previously described.^{52 (link)} PBMCs were isolated from blood of COVID-19 convalescent subjects using human lymphocyte separation medium. Cells were stained and mixed with fluorescent-labeled DNA-barcoded antigens and other fluorescent antibodies, and sorted by FACS with a MoFlo Astrios EQ Flow Cytometer (Beckman). The following antibodies were used to label the B cell subgroups: CD19-PB (BioLegend, Cat 302232), CD27-APC (BioLegend, Cat 302810), CD38-PE (BioLegend, Cat 303506). CD19⁺ CD27⁺ memory B cells and CD19⁺ CD27^high CD38^high plasma B cells were sorted for further scRNA-seq.

For the validation of protein biomarkers, isolated exosomes were incubated for 40 min with one of the following primary antibodies: Glut-1 (Invitrogen, MA5-31960, SA0377), GLYP-1 (Invitrogen, PA5-86043, polyclonal) and ADAM10 (MyBioSource, MBS435195, polyclonal). The stained exosomes were subsequently incubated in the dark for 40 min with the BV421 conjugated secondary antibody (BD Horizon, 565014, polyclonal), in addition to anti-CD63 (Biolegend, 353008, H5C6) and anti-CD81 antibodies (Biolegend, 349512, 5A6). Data was acquired by a MoFlo Astrios EQ Flow Cytometer (Beckman Coulter). Control samples included an unstained exosome sample and several fluorescence-minus-one samples (Table S1). Flow cytometry data were analyzed using FlowJo analysis software.