Macs multistand

To extract electron-dense granules, clMagR/clCry4-transfected bacteria (under exogenous iron supply condition) were harvested, washed, and resuspended in PBS and then fragmented using an ultrasonic cell disruptor (Scientz-II D, amplitude 15%, pulse 5 s on and 2 s off). Subsequently, electron-dense granules were separated from cell debris using gradient centrifugation, washed and resuspended in Milli-Q water to remove residual PBS. The biosynthesis of materials was isolated from the suspension using the MACS LD separation column, the QuadroMACS Separator, and the MACS MultiStand (Miltenyi Biotec Inc) according to the manufacturer’s instructions. The resulting materials were placed on carbon-coated copper grids and dried. Bright-field scanning transmission electron microscopy (BF-STEM), dark-field STEM (DF-STEM), high-angle annular dark-field STEM (HAADF-STEM), energy-dispersive X-ray spectroscopy (XEDS) element mapping, selected area electron diffraction (SAED), and high-resolution TEM (HRTEM) were carried out on FEI Talos F200X TEM at an operating voltage of 200 KV. All micrographs were analyzed using DigitalMicrograph software (Gatan Microscopy Suite, Version 3.42.3048.0) and ICDD PDF-4 + 2009 software (The International Centre for Diffraction Data, ICDD; Powder Diffraction File, PDF). The fast Fourier transform (FFT) was performed using ImageJ, v1.53a software.

TECs were isolated from fresh human GBM (Supplementary Table 1) as described previously^{24 (link)} with slight modification. The tissues were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation) and collected using 40-µm diameter cell strainers (352349, Falcon). CD31⁺ cells were sorted using CD31 MicroBeads with LS Columns (130-042-401) and the MACS MultiStand (No 14281) from Miltenyi Biotec. The TECs were propagated on attachment factor in EC medium supplemented with Bac-off, amphotericin B and penicillin/streptomycin. Cells were harvested with trypsin for passaging and sorted for CD31⁺ expression every two passages. They were not used for experiments after passage 10. Six isolates of normal primary human brain microvascular ECs were purchased from Cell Systems (ACBRI 376, ACBRI 422, and ACBRI 623), Abm (T5458), Cell Biologics (H-6023) and Neuromics (HECO2). NECs were propagated on attachment factor in EC medium as described above for the TECs.
For organotypic culture, GBM tumors were cut into small pieces (~4 mm in diameter) and cultured in Matrigel with neurobasal medium supplemented with EGF (1 ng/ml) and bFGF (1 ng/ml). Tumors were fixed with 10% formalin and embedded in paraffin. In all experiments, cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

The CD4⁺ and CD8⁺ T cells were isolated from CD3⁺ T cells using CD4⁺ microbeads, human (Miltenyi Biotec Cat #130-045-101), and CD8⁺ microbeads, human (Miltenyi Biotec Cat #130-045-201). Briefly, the cells were labeled with specific microspheres and were passed through MACS LS columns (Miltenyi Biotec Cat #130-042-401) placed on the midiMACS Separator (Miltenyi Biotec Cat #130-042-302) and MACS Multistand (Miltenyi Biotec Cat #130-042-303). Enriched cells trapped in the column were plunged into a fresh collection tube, washed, and resuspended in T cell complete media. Samples were collected before and after isolation for flow cytometric analysis as described above.

Tumor‐infiltrating lymphocytes were isolated by CD45 McroBeads using MACS multistand and MidiMACS separator (both from Miltenyi). Anti‐human HLA‐DR (Pacific Blue), CD80 (FITC), CD86 (PE), CCR7(BV 650 TM), and Lin (BV 510TM) were purchased by CST, USA, and used for identifying maturation of DCs.

Buffy coat blood from a healthy anonymous donor (Croix Rouge, Service du sang, Suarlée, Belgium) was used for monocytes isolation. The formed elements of the blood were separated by a high-density gradient of Ficoll 1.077 g/mL (Lymphosep, VWR L0560-100, Nuaillé, France). Erythrocytes and granulocytes settled at the bottom of the tube while lymphocytes and monocytes remained at the sample–separation medium interface and the platelets were in the supernatant. The cell ring containing the Peripheral Blood Mononuclear Cells (PBMC) was collected. Then, the CD14+ monocytes were isolated from PBMC using CD14+ magnetic microbeads (Miltenyi Biotec, Leiden, The Netherlands). Separation columns (Miltenyi Biotec, Leiden, The Netherlands) were used to isolate the CD14+ monocytes with MACS MultiStand (Miltenyi Biotec, Leiden, The Netherlands) and MiniMACS Separator (Miltenyi Biotec, Leiden, The Netherlands) equipment. Monocytes from PBMC were then cultured in RPMI 1640 like THP1.

Single-cell suspensions of mesenteric lymph lymphocytes were prepared under sterile conditions. The desired cells were labeled with CD45R antigen (CD45RA, Miltenyi, 130-090-494). The labeled positive fraction (CD45RA cells) was obtained with the OctoMACS Separator (Miltenyi, 130-042-109) and MACS Multi-Stand (Miltenyi, 130-042-303).