100 μm nylon mesh

Spleens from individual GFP mice were removed and a single-cell suspension was prepared by passing the tissue through a 100 μm nylon mesh (BD Falcon, Bedford, MA). The cells were washed using RPMI 1640 and the red blood cells lysed using 1× red blood cell lysis buffer (eBioscience, Inc., San Diego, CA) and incubated for 1 min. The cells were washed with RPMI 1640, counted on a Cellometer Auto T4 cell counter (Nexcelom, Lawrence, MA), and resuspended in separation buffer (phosphate-buffered saline, pH 7.2, 0.5% bovine serum albumin, and 2 mM EDTA) for cell sorting.

Unnecessary adipose tissue was obtained from a 61-year-old male patient who had previously provided informed consent and underwent plastic surgery. hASCs were isolated using a method described previously [25 (link)]. After washing extensively with phosphate-buffered saline (PBS), the adipose tissues were cut into small pieces and incubated with 3 volumes of 0.1% collagenase (Sigma-Aldrich, St. Louis, MO, USA) solution with constant shaking at 40 °C for 40 min. After adding DMEM containing 10% FBS (Hyclone, Logan, UT, USA) and antibiotics (complete medium), the tissue was centrifuged at 400×g for 3 min. After removing cellular debris through a 100-μm nylon mesh (BD Falcon, Bedford, MA, USA), the cells were incubated in complete medium in a dish. The primary hASCs were cultured for 4–5 days until they reached confluence. These cells were defined as passage “0”. For all experiments, cells from passages 7–9 were used.

The study was approved by the Ethics Review Board of Kansai Medical University in accordance with the ethical guidelines of the Helsinki Declaration of 1975 (approval code: 2006106). All specimens were collected and used with informed consent from the volunteer donors. Adipose tissue was obtained from the patient through plastic surgery. Briefly, the adipose tissues used in this study were resected from fat mass, and not using liquid suction. The abdomen was defined as the donor site, where adipose tissues from disposed tissues from skin graft operations were extracted. hASCs were isolated using a method described previously [7 (link),12 (link),13 (link)]. After extensive washing with phosphate-buffered saline (PBS), the adipose tissues were cut into small pieces and then incubated with 3 volumes of 0.1% collagenase (Sigma-Aldrich, St. Louis, MO) solution with constant shaking at 40 °C for 40 min. After adding Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and antibiotics, it was centrifuged at 400× g for 3 min. After removing cellular remains through a 100 μm nylon mesh (BD Falcon, Bedford, MA, USA), the cells were incubated with DMEM containing 10% FBS and antibiotics in a dish. The primary hASCs were cultured for 4 to 5 days until they reached confluence. For all experiments, cells from passage 7 through 9 were used for the culture.

Spleens from individual MCAO-treated mice were removed and a single-cell suspension was prepared by passing the tissue through a 100 μm nylon mesh (BD Falcon, Bedford, MA). The cells were washed using RPMI 1640 and the red blood cells lysed using 1× red blood cell lysis buffer (eBioscience, Inc., San Diego, CA) and incubated for 1 min. The cells were then washed with RPMI 1640, counted on a Cellometer Auto T4 cell counter (Nexcelom, Lawrence, MA), and resuspended in staining medium [PBS containing 0.1% NaN3 and 1% bovine serum albumin (Sigma, IL)] for flow cytometry. The brain was divided into the ischemic (right) and non-ischemic (left) hemispheres, digested for 60 min with 1 mg/ml Type IV collagenase (Sigma Aldrich, St. Louis, MO) and DNase I (50 mg/ml, Roche Diagnostics, Indianapolis, IN) at 37°C with intermittent shaking. Samples were mixed with a 1 ml pipette every 15 min. The suspension was washed 1× in RPMI, resuspended in 80% Percoll overlayed with 40% Percoll, and centrifuged for 30 min at 1600 RPM. The cells were then washed twice with RPMI 1640 and resuspended in staining medium for flow cytometry.