Ma900 multi application cell sorter

Erythroid cell differentiation was assessed using antibodies against glycophorin A (GPA), integrin α4 chain, band 3, and 7AAD with a BD FACSCanto™ II flow cytometry system (BD Biosciences, Franklin Lakes, NJ, USA) as described before [20 (link)]. Phosphatidylserine (PS) exposure was measured using an Annexin V-FITC kit following manufacturer’s recommendations (BD Biosciences, Franklin Lakes, NJ, USA). The anti-ABCG2 PE antibody was purchased from Biolegend (San Diego, CA, USA). K562 ABCG2 cells expressing low, medium, and high levels of ABCG2 were sorted by flow cytometry using an MA900 Multi-Application Cell Sorter (SONY, Tokyo, Japan).

Four to twenty milliliters of peripheral blood were drawn from each donor into Vacutainer^® K2E (EDTA) Plus Blood Collection Tubes (BD Biosciences, Plymouth, UK). Blood samples were subjected to immediate preparation. In cases where cell enrichment was not performed directly from blood, mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich). Serum samples were collected on the same time into Vacutainer^® SST™ tubes (BD Biosciences, Plymouth, UK). Alternatively, the plasma fraction was isolated from EDTA blood. Bronchoalveolar lavage (BAL) was passed via 70 mcm cell strainer and lymphoid cells were isolated using anti-human CD45 microbeads (Miltenyi Biotec) and live cells were further sorted using a MA900 Multi-Application Cell Sorter (Sony Biotechnology). Both the serum and plasma samples were stored at −20 °C until further use.

Immune cell populations after magnetic-activated cell sorting (MACS) isolation were determined. MA900 Multi-Application Cell Sorter (Sony Biotechnology) was used to detect the differently stained cells. For each sample, a stained and an unstained control were analyzed. The cells were transferred to U-bottomed flow cytometry (FACS) tubes and were incubated with human FcR Block (Miltenyi Biotec, #130-059-901) for 10 min at 4°C. Viability stain took place for 30 min at 4°C using LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit (Invitrogen, #L34959). Staining was performed as follows: 5 µL of CD19 Antibody (BD Horizon™ BB515 Mouse Anti-human CD19, #564456), 20 µL of CD3 Antibody (BD Pharmingen™ PE Mouse Anti-Human CD3, #555340), 5 µL of CD56 Antibody (BD Pharmingen™ PE-Cy™ 7 Mouse Anti-Human CD56, #557747), 5 µL of CD14 Antibody (BD Horizon™ BV421 Mouse Anti-Human CD14, #565283), 5 µL of CD45 Antibody (BD Horizon™ BV786 Mouse Anti-Human CD45, #563716) in 110 µL FACS buffer [PBS + 0.5% FCS (Biological Industries) + 2 mM EDTA (Sigma-Aldrich, #E7889)] per sample. After 15–20 min at 4°C, the samples were washed, centrifuged, and suspended for measurement in FACS buffer. For each sample, 100,000 events were measured. Data were analyzed using the FlowJo_v10.7.1 software (FlowJo, LCC).

Single nuclei were obtained from flash-frozen tissues using sectioning and mechanical homogenization as previously described^{2 (link),54} . Slices of 5–10 mm thickness from frozen tissue were first sectioned with a cryostat in a 50-μm thickness section. All sections from each sample were homogenized using a 7 ml glass Dounce tissue grinder set (Merck) with 8–10 strokes of a loose pestle (A) and 8–10 strokes of a tight pestle (B) in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl₂, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl⁻¹ RNaseIn, 0.2 U μl⁻¹ SUPERaseIn and 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered through a 40 μm cell strainer (Corning). After centrifugation (500g, 5 min, 4 °C), the supernatant was removed and the pellet was resuspended in storage buffer (1× PBS, 4% BSA and 0.2 U μl⁻¹ Protector RNaseIn). Nuclei were stained with 7-AAD viability staining solution (BioLegend), and positive single nuclei were purified by FACS using a MA900 Multi-Application Cell Sorter (Sony) and its proprietary software (Cell Sorter v.3.1.1) (Supplementary Fig. 7). Nuclei purification and integrity were verified by microscopy, and nuclei were further processed for multiome paired RNA and ATAC-seq using Chromium Controller (10x Genomics) according to the manufacturer’s protocol.

Embryonic day 16.5 thymuses and skins, and day five post birth skins were digested in 1 mg/mL collagenase (Wako, 037-17603) in DMEM (Nacalai Tesque, 08459-64) at 37 °C for 15 min, and then in 0.25% trypsin in 1 mM EDTA-PBS at 37 °C for 5–10 min. After centrifuging, cell pellets were resuspended to Feeder Medium and filtered through a 37-µm mesh. Samples were stained with Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (1:100, Biolegend 118212) and PerCP anti-mouse CD45 (1:100, Biolegend 103130) at 4 °C for 30 min. An MA900 Multi-Application Cell Sorter (Sony) was used to collect Epcam-positive and CD45-negative cells (10,000 cells/batch).
Since the E16.5 thymic samples were small in size and composed of a low number of thymic epithelium cells, five thymuses were pooled together as one batch.

OT-1 splenic lymphocytes were isolated using the splenic lymphocytes isolation kit (Solarbio, P8860), following the manufacturer’s instructions. After that, splenic lymphocytes were stained with Anti-CD3-APC (Biolegend, 100326) and Anti-CD8-BV711 (Invitrogen, 407-0081-82). Then, OT-1 CD8 + T cells were sorted and collected using MA900 Multi-Application Cell Sorter (SONY, Japan). The sorted cells were then expanded and activated for five days in RPMI 1640 containing 10% FBS, 5ug/ml IL-2 (Peprotech, 200-02-50), and 1ug/ml OVA257-264 peptide (Genscript, RP10611)^{106 (link)}. After activation, the purity of CD8 + T cells was tested. Then, OT-1 CD8 + T cells and LLC-OVA target cells were co-culture at a 2:1 ratio overnight.
After the co-culture, the suspended CD8 + T cells and apoptotic unattached cells were gently washed away using PBS. Afterward, the surviving adherent tumor cells were fixed and stained with crystal violet solution (Beyotime, C0121). Images were captured and quantification was performed using Image J software. In addition, a flow cytometric analysis was carried out on the cells after the co-culture to determine the number and proportion of surviving tumor cells. DAPI was used to identify dead cells, and the surviving tumor cells were counted by identifying cells with negative DAPI staining and positive mCherry expression.