Magnetic cell sorter

The cell suspension of thymic tissue was prepared by enzymatic digestion with collagenase D (Roche) and DNase I (Roche) as described [9] (link). For TEC isolation, CD45⁻ cells from total cells of thymus tissues were first enriched by depleting CD45⁺ cells using a magnetic cell sorter (Miltenyi Biotec). Consequently, these enriched CD45⁻ cells were stained with APC-conjugated CD45 (SB1) and FITC-conjugated antibody specific for I-A^b or I-A^d, and the stained CD45⁻I-A⁺ cells were isolated to be TECs by FACS Aria (BD Bioscience). To detect the proportion of isolated TECs expressing a medullary TEC (mTEC) marker, CD45⁻I-A^b+ or CD45⁻I-A^d+ TECs cells were stained with biotinylated-Ulex Europaeus Agglutinin 1 (UEA1) accompanied with streptavidin-PE. For isolation of TECs from FTOC, the cultured thymic lobes were washed and subjected to enzymatic digestion as described above for adult thymus.

Human MNCs were isolated from blood samples by Ficoll-Paque PLUS (GE Healthcare, Chicago, IL, USA) and incubated with CD14 microbeads. CD14+ cells were eluted from the positive selection column of Magnetic Cell Sorter (Miltenyi Biotec, Germany). CD14- cells were incubated with CD4 microbeads, and CD4+ cells were eluted from the column. Mouse spleens were homogenized by using syringe plunger and mesh strainer. Mouse MNCs were further incubated with PE-Cy5 anti-CD4 (BD Pharmingen, San Diego, CA, USA) or FITC anti-CD19 (BD Pharmingen), and sorted by Moflo XDP Cell Sorter (Beckman Coulter, Mountain View, CA) to obtain CD4+ or CD19+ cells. Purity of cell subpopulation was confirmed to be up to 95 % by flow cytometric analyses. Human urine cells were isolated from urine specimens by centrifugation and washing procedures to obtain cell pellets [37 (link)]. After removing capsules, mouse kidneys were minced into tiny pieces to obtain cortex tissues, followed by incubation with digestion buffer with collagenase (Sigma-Aldrich), and centrifuged to collect cell pellets [38 (link)].

The human glioblastoma cell line U87/MG and human NK cell line NK92-MI were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). U87/MG cells were cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Hyclone). NK92-MI cells were cultured in stem cell growth medium (CellGro, Freiburg, Germany) supplemented with 2% exosome-depleted human serum (ultracentrifuged at 100,000 × g for 18 h) and 1% penicillin–streptomycin, at 37°C in 5% CO₂. U87/MG cells were transfected with a recombinant retrovirus containing a plasmid that showed enhanced expression of firefly luciferase (effluc) and thy1.1 genes, driven by a long terminal-repeat promoter (Retro–LTR–effluc–thy1.1). Thy1.1-positive cells were sorted from U87/MG cells expressing both effluc and thy1.1 genes using a magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany). Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to confirm the expression of effluc mRNA and protein, respectively. Established stable cells expressing both effluc and thy1.1 genes were referred to as U87/MG/F cells.

After 8-week-old male C57BL/6N mice were sacrificed, BMCs from femurs and tibia were collected and cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal bovine serum supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin containing either 10 ng/mL GM-CSF (R&D Systems, Minneapolis, MN, USA) or 10 ng/mL M-CSF (R&D Systems, Minneapolis, MN, USA). On day 7, adherent cells were harvested. F4/80⁺ macrophages were stained with an FITC-conjugated rat anti-mouse F4/80 antibody (eBioscience, San Diego, CA, USA) and anti-FITC MicroBeads (Miltenyi Biotec, Gladbach, Germany), and were enriched using a Magnetic Cell Sorter (Miltenyi Biotec, Gladbach, Germany). Expression of the CD11b, CD11c and F4/80 on GM-CSF or M-CSF treated-BMCs was assessed after 7 days in culture using the following antibodies: CD11b-FITC (BD Biosciences, San Jose, CA, USA), CD11c-PE (eBioscience, San Diego, CA, USA) and F4/80-APC (Biolegend, San Diego, CA, USA). Rat IgG2a kappa-APC (eBioscience, San Diego, CA, USA) was used as isotype control. Then, the cells were analyzed by flow cytometry (BD FACSVerse, BD Biosciences, San Jose, CA, USA) using FACSuite software. Data were analyzed using FlowJo software (Tree Star).