70 μm nylon mesh

Tumour tissues were washed with PBS three times, cut into small pieces with scissors, and then minced completely using sterile scalpel blades. The minced tumour pieces were incubated with ultrapure collagenase IV (Worthington Biochemicals, Freehold, NJ, USA) at 37 °C for 1 h, with manual dissociation every 15 to 20 min by pipetting. Single cells were filtered through a 70-μm nylon mesh (Corning) and washed with PBS supplemented with 2% FBS. Cells were stained with specific antibodies according to the manufacturer’s instructions. The antibodies including anti-mouse CD45 (HI30), CD4 (RM4-5), CD11b (ICRF44), GR-1 (RB6-8C5), FOXP3 (236 A/E7), anti-human CD68 (Y1/82 A), CD163 (RM3/1), CD206 (15-2), mouse Fc block (2.4G2), and human Fc block (TruStain FcX) were purchased from BD Biosciences or Biolegend. Isotype controls were used for all staining. Flow cytometry was performed on a BD FACS Canto-II Flow Cytometer (BD Biosciences), and data were analysed using FlowJo software (version 10.6, Ashland, OR).

Spleens were processed to produce single-cell suspensions by manual disaggregation, erythrocyte lysis using an ammonium-chloride-potassium lysing buffer (Gibco), and passage through a 70 μm nylon mesh (Corning). B cells were purified from these single-cell suspensions using the negative isolation EasySep Mouse B Cell Isolation Kit (catalog 19854, STEMCELL Technologies). The purity of B cells following isolation was confirmed via flow cytometry and greater than 97%. Bregs-CpG were produced by culturing purified B cells in complete medium (RPMI 1640 containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, 1× nonessential amino acids, 100 IU/mL penicillin, and 100 μg/mL streptomycin) with CpG B ODN 1668 (10 μg/mL, Class B, murine TLR 9–specific, InvivoGen) for 3 days. Bregs-TLR were generated on Bregs-CpG by adding LPS (10 μg/mL), PMA (50 ng/mL), and ionomycin (1 μg/mL) on the final day for 5 hours before collection. LPS-B cells were cultured with LPS (10 μg/mL) alone for 3 days. All culture additives were purchased from MilliporeSigma unless noted otherwise.

To visualize blood clots (thrombosis) and bone marrow-derived ECs in the cerebral vessels
of TgNotch3R90C mice, the bone marrow of the transgenic mice ubiquitously expressing
enhanced GFP under the control of the human ubiquitin C promoter (UBC-GFP) was
transplanted into the TgNotch3R90C mice (C57BL/6 background, a gift from Dr Anne Joutel’s
lab). UBC-GFP mice (male, 6–8 weeks old; C57BL/6 background, Jackson Laboratory) were
anesthetized with Avertin (0.4g/kg body weight, intraperitoneally (i.p.); Sigma-Aldrich,
St. Louis, MO, USA). The femur bones were dissected and placed into a dish with ice-cold
sterile Hanks Balanced Salt Solution (HBSS; ThermoFisher Scientific, Pittsburgh, PA, USA).
Bone marrow cells were flushed out with a 25G needle. Cells were gently triturated with a
10 ml pipette, filtered through a 70 μm nylon mesh (Corning, Fisher Scientific,
Pittsburgh, PA, USA) and collected in a 50 ml tube (Corning, Fisher Scientific,
Pittsburgh, PA, USA). Harvested cells were centrifuged and re-suspended with HBSS into
single cell suspension. Cells were transplanted to irradiated TgNotch3R90C mice by tail
vein injection (1×10^{7 (link)} bone marrow cells in 0.6 ml HBSS per mouse).

The in vitro effects of the extracts and adenosine-related compounds (cordycepin, 5′-methylthioadenosine, S-adenosylmethionine guanosine, inosine, adenine, and caffeine) on testicular testosterone production were examined in primary cultures of mouse testicular cells prepared using the method of Yang et al. [25 (link)]. Briefly, the testes of ddY mice (male; age, 7–11 weeks; Japan SLC Inc.) were excised, decapsulated, and incubated in medium 199 (Sigma-Aldrich) containing 0.4% collagenase and 0.1% bovine serum albumin (BSA) at 37 °C for 15 min. The testes were cooled, passed through a 70-μm nylon mesh (Corning Inc.), and centrifuged at 450× g for 15 min at 4 °C. The cells were washed with DME/F-12 medium and seeded in DME/F-12 containing 10% FBS at a density of 3 × 10⁵ cells/well in a 24-well plate (Primaria, Corning Inc.). After incubation for 18–24 h, the cells were washed twice with DME/F-12 and incubated with diluted extracts or compounds in DME/F-12 containing 0.1% BSA for another 2–3 h and the medium was collected and subjected to enzyme immunoassays (EIAs) for the measurement of testosterone. The testes obtained from the experiments described in Section 2.3 were also used without stimulants. The validation of primary culture preparation was performed using hCG as a positive control.

The cauda epididymis was collected and minced with a razor blade in 1 mL PBS. The suspension was filtered through 70-μm-nylon mesh (Corning, Corning, NY). The number of spermatozoa was counted using a hemocytometer.

Ankle joints were collected, diced, and digested with 2 mg/ml dispase and 1 mg/ml collagenase type 1 (Thermo Fisher Scientific, USA) in RPMI 1640 + 10% fetal bovine serum (FBS) for 1 h at 37 °C. The cells were filtered through a cell strainer with a 70 μm nylon mesh (Corning, USA) and washed with RPMI 1640 + 10% FBS. Cells were then stained with a standard panel of immunophenotyping antibodies. A list of all antibodies used in the study is shown in Table S1, including anti-mouse CD11b (1:500) and anti-mouse Ly6G (1:500), for 1 h at room temperature. After staining, cells were suspended with a staining buffer; then, samples were captured using FACS Canto II Cytometry (BD Biosciences, USA), and the data were analyzed with FlowJo software (BD Biosciences, USA).

Bacteria (S. aureus strain SH1000) were homogenized by ultrasound and injected in tail vein of the mice in 100 μl PBS (Gibco by Life Technologies, Karlsbad, California). Bacteria were injected at indicated concentration (cfu/μL). For quantification of renal bacteria, kidneys were dissected and incubated for 40 min at 37°C in RPMI1640 medium (including 0.01 M HEPES; 0.5 mL filtered FBS and 0.4 μg/ mL DNase and 8 μg/ mL collagenase D). GentleMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and sonication for 60 seconds using a Bandelin Sonorex Super RK103 were used for homogenization.
For quantification of hepatic or splenic bacteria these organs were passed through 70 μm nylon mesh (Corning, Corning, New York) and resuspended in PBS before sonification. The samples were incubated for 24 h at 37°C on LB agar plates and colonies were counted. S. aureus was verified by MALDI-TOF.