Tergitol

FACS‐sorted 1 × 10⁴ naïve CD8WT OT‐I, CD8.4 OT‐I (VM), sorted true memory OT‐I T cells, or no T cells were adoptively transferred into Ly5.1 C57Bl/6j mice followed by infection with 5,000 CFU Lm‐Q4H7. The recipient mice were sacrificed on day 3 and 5 post‐infection, and the spleen was homogenized and lysed in PBS with 0.1% Tergitol (Sigma‐Aldrich). 1/20 of splenic lysate was plated onto brain–heart infusion agar (BHI, Sigma‐Aldrich) plates with 200 μg/ml streptomycin and incubated at 37°C. The resulting number of colonies was quantified the following day.

SKBr3 cells were harvested for western blot analysis by trypsinization with 0.25 % trypsin-EDTA (Thermo Scientific). Cells were washed with 1x PBS (Thermo Scientific) and resuspended in cold radioimmunoprecipitation assay buffer (RIPA) 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% Tergitol (Sigma-Aldrich, St. Louis, MO, USA), 0.5% Na-deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4, and 1 × Halt Protease Inhibitor Cocktail (Thermo Scientific). Protein concentration was determined by BCA assay (Thermo Scientific). 4x Laemmli buffer containing 10% 2-mercaptoethanol was added to lysates and boiled at 95 °C for 5 minutes. Lysates were loaded on 8% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad).

The Tergitol-based decellularization protocol was evolved from the TRICOL protocol previously reported by our group [15 (link)]. Briefly, tissues were thawed at RT for 3 h, before initiating the decellularization procedure. Then, they were submerged in 500 mL sterile jars in agitation at 4 °C with proteases inhibitors cocktail for 8 h, afterwards with 1% Tergitol (Sigma) at RT for the next 12 h; hypertonic/hypotonic shocks (8 h each cycle at RT) followed, and finally they were treated with 0.4% sodium cholate (Sigma) for 16 h in the dark at RT. After decellularization, 2 cycles of washing with PBS (1X) were performed. Ethanol (4%, Carlo Erba, Cornaredo, Milano, Italy) and peracetic acid (0.1%, Sigma) solution was used for bioburden reduction. Finally, an endonuclease (Benzonase 25k U, Sigma, E1014) was applied to remove nucleic acids for 48 h at 37 °C.

Mice represents different housing conditions were euthanized (sodium pentobarbital 45mg/kg; intraperitonially) after the behavioural test. Hippocampus region (n = 6/group) was dissected out from the whole brain, and stored at -70°C. The hippocampus tissue from each individual was used to isolate total RNA, protein and Gas Chromatography-Mass Spectrometry (GC-MS) analysis, and feces (n = 6/group) also collected from the same individuals. The whole brain was dissected out (n = 4/group), and immediately processed for immunohistochemistry.
Hippocampus tissue was homogenized in lysis buffer [Tris-Hydrochloride (50mM; pH 7.5; Cat# RM613; HiMedia, India) Sodium chloride (150mM; Cat# 1.93206.0521; Merck, India), Ethylenediaminetetraactetic acid (EDTA) Disodium salt (50 mM; Cat# RM1195; HiMedia, India), Tergitol (NP-40; 0.1% V/V; Cat# MKCD6607; Sigma-Aldrich, India), Dithiotheritol (DTT,1 mM; Cat# MB070; HiMedia, India) Sodium orthovanadate (Na3Vo4, 0.2Mm; Cat# S6508; Sigma-Aldrich, India), Phenylmethylsulfonyl fluoride (PMSF;0.023mM Cat# P7626; Sigma-Aldrich, India)] following the procedure as reported [40 (link)]. Final supernatant was collected, concentration of each samples were measured using Bio-photometer Plus (Eppendorf Inc., Germany) and stored in aliquots at -80°C.