Dynabeads m 450 tosylactivated

Evans blue, methamphetamine (METH) and MLA were purchased from Sigma-Aldrich (St. Louis, MO). Dynabeads M-450 Tosylactivated was purchased from Invitrogen (Carlsbad, CA). Ulex europaeus I (UEA I) lectin and mounting medium with DAPI were purchased from Vector (Buringame, CA). The Gp41 ectodomain peptide (gp41-I90) was prepared as described previously [9 (link), 11 (link)]. A senescence β-Galactosidase Staining Kit (Cat#9860) was bought from Cell Signaling Technology. A Fluoro-Jade B (FJB) staining kit was obtained from Histo-Chemo Inc. All primary antibodies (Ab) were purchased from the commercial sources: a rabbit anti-α7 nAChR Ab from Genescript (Piscataway, NJ); Anti-NF-κB/p65 mAb from Cell Signal Technology (Cell Signal Technology, #3033); an antibody against dimethyl-histone H3 (Lys9) from Millipore (Millipore, cat #.07–212), an anti-mouse CD146 Ab FITC-conjugated and a mouse anti-neuron (NeuN) Ab from eBiosciences (San Diego, CA); a mouse anti-CD44 Ab (sc-7297); a rabbit anti-CD54 Ab (ICAM-1, 250593) from Abbiotec (San Diego, CA); a rabbit anti-β-actin (sc-7210) and an anti-GFAP Ab from Santa Cruz Biotechnology (Santa Cruz, CA); an anti-mouse CD146 Ab FITC-conjugated from Biolegend (San Diego, CA), and a rabbit anti-S100B Ab from BD Biosciences.

Anti-CD3 antibody (145-2X11, R&D systems, Minneapolis, MN, USA) together with recombinant mouse CD155.Fc or the control.Fc (Sino Biological, Beijing, China) or PD-L1. Fc or the control.Fc (R&D systems) or HVEM.Fc (R&D systems) were covalently attached to Dynabeads M450 Tosylactivated (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Anti-CD3 antibody together with control.Fc was used for the control. For each 10⁷ beads, 1 μg of anti-CD3 antibody (20% of total protein) and 4 μg of CD155.Fc, PD-L1.Fc, HVEM.Fc or control.Fc (80%) were used.

PBS with Dynabeads M-450 Tosylactivated (Invitrogen, 14013) was perfused into the mouse aorta; kidney tissues were dissected, minced, and digested in 1 mg/mL collagenase A and 100 U/mL DNase I for 30 minutes at 37°C. Digested mixture was then passed through a 100 μm cell strainer and placed on a magnet for purification. The purity of glomeruli was more than 95% confirmed by microscopy.

To identify the target protein expression profile, mouse glomeruli were isolated as described previously [22 (link)]. In brief, mice were anesthetized and perfused with 8 × 10⁷ Dynabeads M-450 Tosylactivated (Invitrogen, Cat No. 14013) diluted in phosphate-buffered saline (PBS) through the heart. The kidneys were removed and the cortex, medulla, and papilla were separated by dissection. Cortex was minced into 1 mm³ piece and digested in collagenase (Sigma-Aldrich, Cat No. C6885) at 37 °C for 15 min. Then, the tissue was pressed through a 100 μm cell strainer, and the cell suspension was centrifuged for the pellet. The pellet was resuspended in PBS, and the glomeruli containing Dynabeads were gathered using a magnetic particle concentrator and washed at least three times with PBS. The supernatant enriched with proximal tubules was washed and collected for analysis.
Rat glomeruli were isolated as previously described [35 (link)]. Briefly, the renal cortexes of SD rats were minced and gently pressed through a series of sieves (200, 120, and 75 μM), and the glomeruli were then gathered on the last sieve.

Dynabeads M-450 Tosylactivated (Invitrogen) were used to covalently couple monoclonal antibodies. Briefly, beads were incubated in 1 ml of phosphate buffer 0.1 M pH 7.6 with each mAb at 37°C overnight in rotation. Unreacted tosyl groups were blocked by incubation with buffer tris 0.2 M 0.1%BSA pH 8.5 for 30 min at room temperature. Beads were then washed twice with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) 2 mM EDTA pH 7.4 and stored at 4°C. The following monoclonal antibodies were used to couple microbeads to simulate human T cells: anti-human CD3 (BE0001-2, Bio X cell) and anti-human CD3 (produced in house), anti-human CD137 (6B4, mouse IgG1, produced in house), mouse IgG2a (40202, Biolegend), mouse IgG1 (400102, Biolegend) (Supplementary Table 1). In the case of microbeads coupled to CD137L, the CD137L-Fc (Sino Biological) was used (Supplementary Table 2). In the case of microbeads coupled to anti-CD7, the anti-CD7 mAb (395602, Biolegend) was used. The following monoclonal antibodies were used for mAb coated beads to stimulate mouse T cells (Supplementary Table 3): antimouse CD3 (100238, Biolegend), antimouse CD137 (BE0239, Bio X cell), antimouse CD137 (3H3, produced in house), rat IgG2a (400533, Biolegend), rat IgG2b (400637, Biolegend). Details of concentrations for antibody coupling are provided in the tables.

Isolation was performed based on the magnetic bead method [47 (link)] with minor modifications. In brief, anaesthetized mice undertook a midline thoraco-abdominal incision; two ligations were placed, the first one closing the abdominal aorta together with the inferior cava vein below the branching point of the renal vessels, the other one closing the coeliac trunk together with the superior mesenteric artery. Both kidneys were perfused with 100 µL (= 4 × 10⁷) Dynabeads M-450 Tosylactivated (Invitrogen/Life Technologies AS, Oslo, Norway) in ice-cold HBSS (Ca²⁺, Mg²⁺) via a polyethylene tubing extension on top of a 24G needle, inserted and secured into the upper abdominal aorta, while volume outflow was ensured by opening the distally ligated vena cava inferior (VCI). ECM extraction was performed according to a protocol established before [7 (link)].

Mice were anesthetized and laparotomy–thoracotomy was performed. Intracardiac perfusion of Hank’s Balanced Salt Solution (HBSS) (#14025-076, Gibco Laboratories, Gaithersburg, MD, USA) was performed to exsanguinate the mice followed by perfusion with Dynabeads™ M-450 Tosylactivated (#14013, Invitrogen, Vilnius, Lithuania) diluted in Tris (0.2 M, pH 8.5; 0.1% BSA). Kidneys were harvested and then homogenized on ice. Homogenates were digested in HBSS with 10 mg/mL Collagenase type II (#17101-015, Gibco Laboratories) and DNase (#04716728001, Roche Diagnostics, Mannheim, Germany) for 1 h on an orbital shaker (37 °C, 160 rpm). Digested tissue was passed through a 100 µM cell strainer and washed with HBSS. Filtrate was centrifuged at 200× g for 5 min and the resulting pellet was resuspended in HBSS. Suspension was placed on a DynaMagnet (#12303D, Invitrogen, Oslo, Norway) and subjected to serial washes with HBSS to yield purified glomeruli.