Hank s balanced salt solution

For the characterization of gross morphology, the identified scPVS was isolated from the abdominal hypodermis region using a microscissor (blade 5 mm) and transferred into a drop of Hank's balanced salt solution (Sigma, St. Louis, MO, USA) on a slide glass. After the sample was completely air-dried at room temperature for 1–3 min, the slide glass was immersed 10 times in and taken out of dye Solution 1 for 10 s (~1/s). Additional staining with Solutions 2 and 3 was repeated using the same process. The sample was then placed in a drop of phosphate buffer solution (pH 7.2) for 20 s, washed by shaking it 10 times in distilled water for 20 s, air-dried for 3–5 min, and finally mounted with Canada balsam (Sigma). The PVS slice preparation was performed according to the previous method [6 (link)]. To confirm the DNA and RNA contents of the scPVS, the sample was kept immersed in a 0.1% acridine orange solution for 15 min, and a digital photograph was then taken using a confocal laser-scanning microscope at the wavelength of the acridine orange [19 (link)]. To verify the MCs in the scPVS, the sample was immersed in a 1% toluidine blue solution for 3 min [20 (link)].

Primary microglia cultures were prepared from cerebral cortices of C57BL/6J mice. Briefly, mice were sacrificed, and brains were carefully dissected, chopped, triturated, and freed from meninges in Hanks' balanced salt solution (Sigma-Aldrich, USA). Forebrains were gently minced, dissociated, resuspended in DMEM/F-12 medium (Gibco, Shanghai, China), and filtered by passing through a 70 mm cell strainer (Sigma). Cells were collected by centrifugation (1,000 × g, 10 minutes at 4°C) and resuspended in DMEM/F-12 containing 10% (v/v) fetal bovine serum (FBS, Gibco, Shanghai, China), 100 U/ml penicillin, and 100 mg/ml streptomycin and cultured on Poly-D-lysine coated 75 cm² cell culture flask in a humidified incubator with 5% CO₂ at 37°C. After 12-14 days in culture, floating microglia were harvested from mixed glia (astrocyte/microglia) cultures and reseeded into cell culture plates. On the next day, nonadherent cells were removed by changing the media, and after 1 h, cells were used for subsequent experiments.

Hydrogen peroxide, ferric sulfate, ferric chloride, catalase, the Hydrogen peroxide assay kit, and the phosphorothioate-modified dinucleotides dGsA and dGA were purchased from Sangon Biotech (Shanghai) Co., Ltd. Trimethoprim, thymine, α-(4-pyridyl-N-oxide)-N-tert-butylnitrone (POBN), diethylenetriaminepentaacetic acid (DTPA), Hanks’ Balanced Salt Solution (HBSS, without phenol red, calcium chloride and magnesium sulfate), threonine, NAD⁺ and Chelex-100 were purchased from Sigma. The DNeasy Tissue kit was purchased from QIAGEN. The Cycle-Pure Kit, Bacterial DNA Kit, and Gel Extraction Kit were purchased from OMEGA Bio-Tech. Luria-Bertani (LB) broth contained 10 g/L of BactoTryptone, 5 g/L yeast extract, and 10 g/L sodium chloride unless otherwise noted. The modified marine broth medium 2216E contained 5 g/L tryptone, 1 g/L yeast extract, 0.1 g/L FePO₄, and 34 g/L sodium chloride. K medium contained A salts, 0.2% glucose, 1 mM MgCl₂, 0.5 mM amino acids and 5 mg/L thiamine. The media were supplemented with the following concentrations of antibiotics (unless otherwise noted): ampicillin (Amp) 100 μg/ml and chloramphenicol (Cml) 12.5 μg/ml. Solid medium was supplemented with 1.5% (w/v) agar-A.

Colons were obtained from mice fed ad libitum and from mice after 36 h fasting. ECs were isolated from the colon and treated with 10 mM EDTA in Hank’s Balanced Salt Solution (Sigma Aldrich). Forty thousand cells/well in a 96-well half plate were cultured in 20% Matrigel (Corning, Tokyo, Japan) in a culture medium containing D-MEM/Media 199 (mixture of 4:1, Sigma and Lonza, Tokyo, Japan), 0.01 M HEPES, 1 × antibiotic-antimycotic (Gibco), 50 µg/ml gentamycin (Gibco) supplemented with 10% Hyclone Cosmic Calf serum (GE Healthcare), 50 ng/ml mEGF (Peprotech, Rocky Hill, NJ), 1 µg/ml hydrocortisone (Sigma-Aldrich), 2 µg/ml Transferrin (Sigma-Aldrich), and 5 nM sodium selenite (Sigma-Aldrich) in 5% CO₂ and 5% O₂ atmosphere. To study the effects of organic acids, lactate, butyrate, or acetate (10 mM at final concentration, pH was adjusted to 7 with NaOH, Wako, Japan) was added to the culture medium. Fresh medium (0.05 ml) with/without organic acids was added to the culture every 4 days (day 4, 8 and 12). After day 4 of culture, the number of colonies containing more than 10 living cells/well was counted everyday with a inverted microscope (IX71, Olympus, Tokyo, Japan). Cultures were performed in triplicate and data were presented as the mean ± SD, unless otherwise indicated in the legend.

To assess cell death, cells were washed twice in standard media and then seeded in duplicate at 1.5 × 10⁴ cells/mL in 96-well U-bottom plates. Cells were either untreated, treated with vehicle control (DMSO), or treated with varying concentrations of JAK inhibitors. After a 72 h incubation at 37 °C with 5% CO₂, cells were washed once with HANK’s Balanced Salt solution (Sigma-Aldrich, CAT#H9394) supplemented with 5 mM calcium chloride (Sigma-Aldrich, CAT#C1016) and 1% HEPES (Sigma-Aldrich, CAT#H0887) (binding buffer). The percentage of cell death was determined by staining with 2% annexin-V-PE (BD Biosciences, CAT#556421) and 0.25% Live/Dead Fixable Aqua Dead Cell Stain (Invitrogen, CAT#L34957) (aqua dead cell stain) in 20 µL of binding buffer for 1 h in the dark. Cells were washed and analyzed on the BD FACSCanto II (BD Bioscience); then, flow cytometry data were analyzed and plotted using FlowJo analysis software v10 (FlowJo). The gating strategy used to determine the percentage of viable cells is shown in Supplementary Fig. 10b. The percentage of viable cells was normalized to the first and last means of each dataset and plotted using GraphPad Prism v8 (GraphPad Software). Non-linear regression curves were fit to appropriate data to determine median lethal dose (LD₅₀).

Pancreatic islets were isolated as previously described (Foda et al, 2020 (link)). Briefly, pancreatic islets were isolated by perfusing the pancreas via the common bile duct with collagenase P solution (0.5 units/ml). The inflated pancreata were incubated for 16 min at 37°C, agitated, and washed with Hank’s balanced salt solution (Sigma-Aldrich) plus 2% fetal bovine serum. Islets were hand-picked from the pancreas digestion slurry and dissociated in non-enzymatic cell dissociation buffer. Islet cell suspensions were resuspended in RPMI and incubated at 37°C for 1 h. Final islet single cell suspensions were washed and resuspended in RPMI. Spleens were harvested and mechanically processed with a 70 μm filter to obtain a single-cell suspension. Red blood cell lysis was performed using ACK lysing buffer (Lonza). Splenocytes were enriched for CD8 T cells using magnetic bead-based negative selection (CD8 mouse T-cell isolation kit; Miltenyi Biotec). Final spleen single-cell suspensions were washed and resuspended in RPMI. Islets and spleens were each pooled from 10 mice for both scRNA-seq experiments.