Galios

Exosomes were isolated from the culture medium containing hEpi AD–MSCs. After the cells reached 80% confluence, the medium was collected every 2 d, until 180 mL medium was obtained, which was centrifuged at 300× g for 10 min to discard cellular debris. The supernatant was filtered using the tangential flow filtration system (TFF system; Pall Corporation, Port Washington, NY, USA). The feed flow rate was set at 120 rpm. The cell culture medium was concentrated to 3 mL (60 times) using the TFF system. Expression of exosome surface markers was determined by bead-based flow cytometry (FACS; Galios, Beckman Coulter, Brea, CA, USA) using antibodies against CD63 (BioLegend, 353003) and CD81 (BioLegend, 349,505) as previously described [30 (link)]. To analyze exosome surface-specific markers, exosomes were mixed with 4% aldehyde/sulfate latex beads (Thermo Fisher Scientific, Rockford, IL, USA). Transmission electron microscopy (TEM; HT7700, Hitachi, Japan) was used to observe morphological characteristics of the exosomes. Exosomes were adsorbed on a formvar carbon-coated copper grid (Ted Pella Inc., Redding, CA, USA), fixed with 2% paraformaldehyde for 10 min, and then dried. Particle size distribution and concentration were determined by NTA (Nanosight NS300, Malvern Panalytical, Worcestershire, UK) following the manufacturer’s instructions.

All females were in estrus at sacrifice, as determined by examination of cells from vaginal lavage. Whole inguinal (#4) mammary glands were dissected and digested at 37 °C for 4 h in 1 mg/mL collagenase B (Roche) and 0.5 mg/mL hyaluronidase (Sigma Aldrich) with gentle shaking. Dissociated cells were washed and filtered through a 50-μm cell strainer before incubating with antibodies. All antibodies for flow cytometry were purchased from BioLegend. Antibodies to CD31, CD45, and TER119 were conjugated to Pacific Blue. Additional antibodies used were Alexa-fluor 488 anti-CD24, APC anti-CD29, and PE anti-CD61. Samples were analyzed on a Galios (Beckman Coulter). Analysis was performed with Kaluza Analysis Software (Beckman Coulter).

This study was approved by the Institutional Review Board of Yeungnam University Medical Center in Daegu, Korea (IRB No. 2017-07-032), and informed consent was obtained from all the patients. hEpi AD–MSCs were isolated from human epidural fat as previously described [28 (link),29 (link)]. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 1.0 g/L glucose (DMEM; Gibco, Carlsbad, CA, USA) containing 10% exosome-free fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin–streptomycin (P/S; Gibco, Carlsbad, CA, USA) and incubated at 37 °C with 5% CO₂. hEpi AD–MSCs at passage 5 to passage 10 were used for further experiments. The expression of stem cell markers on these cells was determined by flow cytometry (FACS; Galios, Beckman Coulter, Brea, CA, USA) using the CD73-PE (BioLegend, San Diego, CA, USA, 344004), CD90-PE (BioLegend, 555596), CD105-PE (Bio-Rad, Hercules, CA, USA, MCA1557), CD14-PE (Bio-Rad, MCA1568), CD34-FITC (BioLegend, 343504), and CD45-FITC (BioLegend, 555482) antibodies.

Cell surface antigens were stained in the dark at 4 °C with antibodies diluted in PBS that contained 0.5% bovine serum albumin. Cells were analyzed using a Galios instrument (Beckman Coulter). Dead cells (DAPI⁺) were excluded from the analysis. B cells, T cells, dendritic cells, and macrophages with the B220⁺, CD3⁺, CD11c⁺, or CD11b⁺ genotype were purified (>95%) from Nfkbiz^fl/Δ or Nfkbiz^fl/Δ Mb1^cre/+ mice using a Aria II cell sorter (BD Biosciences).

Analysis of cell surface SiaS expression was performed by FCA. Cells were collected with a cell scraper, washed twice with PBS, and blocked with 5 mM EDTA and 0.5% BSA at 4 °C for 30 min. They were incubated with 3G9 (10 µg/mL) at 4 °C for 1 h, and washed twice with PBS. mAb.2G9 (10 μg/mL) was used as an isotype control. The cells were incubated with Alexa Fluor 488-labeled goat anti-mouse (IgG + IgM) (2 µg/mL) at 4 °C for 30 min. After washing twice with PBS, the cell surface fluorescence was analyzed using a Galios flow cytometer (Beckman Coulter, Brea, CA, USA). The collected data were analyzed using the Kaluza software (Beckman Coulter). The proportion of the 3G9 epitope-positive to negative cells was analyzed.

EAT was collected and cut into small pieces. After filtering through a 40-μm strainer, tissues were centrifuged and resuspended in RPMI(−) medium. The dissociated SVF was washed twice with PBS, incubated for 10 min in erythrocyte-lysing buffer, and washed again with PBS. Cells were stained with the following antibodies and incubated for 30 min at 4 °C in the dark: CD11c (BioLegend, San Diego, CA, USA) and F4/80 (BD Biosciences, Franklin Lakes, NJ, USA). Staining was analysed by fluorescence-assisted cell sorting (Galios; Beckman Coulter, Tokyo, Japan) using FlowJo 7.6.3. software (Tree Star, Ashland, OR, USA).