Tuberculin syringe

The CST (Human CgA_352−372: SSMKLSFRARAYGFRGPGPQL) (Mahata et al., 2010 (link)) was used (Biopeptide Co., Inc., San Diego, CA, USA), and the peptide was injected (i.r.) at 1.5 mg/per kg body weight per day for 6 days. Saline (0.9%) was injected in the control group. Mice were anesthetized using isoflurane (Abbott, Toronto, ON, Canada). PE-90 tubing (10 cm long; ClayAdam, Parisppany, NJ, USA), which was attached to a tuberculin syringe (BD, Mississauga, ON, Canada), was inserted 3.5 cm into the colon. The dose was determined according to our previous published study (Rabbi et al., 2014 (link)).

The hCTS (hCHGA_352–372: SSMKLSFRARAYGFRGPGPQL) was used (Biopeptide Co., Inc., San Diego, CA, USA), and the peptide was injected intrarectally (i.r.) at 1.5 mg/per kg body weight per day for 7 days. Saline (0.9%) was injected into the control group. Mice were anesthetized using Isoflurane (Abbott, Toronto, ON, Canada). PE-90 tubing (10 cm long; ClayAdam, Parsippany, NJ, USA), which was attached to a tuberculin syringe (BD, Mississauga, ON, Canada), was inserted 3.5 cm into the colon. The dose was determined according to our previous published study (31 (link)).

Surgical instruments including Castroviejo scissors, uniband LA-1 micro point scissors, serrated jaw MF-2 micro forceps, fully curved micro forceps MF-3, straight micro forceps soldering tweezers, insertion/extraction tweezers and anti-wicking tweezers were purchased from Cedarlane labs – Canada. Surgical sutures including 5-0 Perma Hand Silk Black 1X18’’ PS-3, 5-0 Perma Hand Silk Black 2X60’’ no needle and 4-0 Perma Hand Silk Black 1X18’’ G-3 were purchased from the Ethicon – USA. Diethylstilbestrol (DES) was purchased from Sigma – USA and prepared in mineral oil (Sigma-Aldrich, USA). GIMA 2mm diameter biopsy punch (GIMA, UK) was used to divide the endometrial layer into 2mm pieces. An 18G syringe needle (BD, USA) attached to a tuberculin syringe (BD, USA) was used to deliver endometrial specimens into the peritoneal cavity. Banamine (Merck, USA) solution was used every 12 hours for 2 two days to alleviate the pain during post-surgical care period. DMEM/F12 medium was used for preparation of endometrial transplant tissue (Sigma-Aldrich, USA). Skin incisions were closed with a Reflex Clip Applier (World Precision Instrument, USA) and clips were removed using Reflex Clip Removing Forceps (World Precision Instrument, USA).

Germ-free 129S6/SvEv mice were mono-colonized with either wild-type (WT) or ΔprtX strains of L. acidophilus NCFM, as described above. On the day of the assay, mice were denied access to food but allowed access to water ad libitum for 3 h, after which 150 μl of 3–5 kDa fluorescein isothiocyanate (FITC)-dextran at a concentration of 80 mg/ml was administered to each mouse using an oral gavage needle. Two hours after administration of the FITC-dextran, blood was collected using a 1 ml tuberculin syringe (BD) and stored in Microtainer serum separator tubes (BD). Blood was placed in the dark for 1 h and allowed to clot, after which the serum was separated via centrifugation (13,000 rpm, 4°C). Fluorescence in the serum was measured using a FLOUStar Optima Microtiter plate reader (BMG Technologies) with the excitation filter set for 490 nm, the emission filter set for 520 nm, and a 1250 gain setting for fluorescence intensity. FITC-dextran fluorescence was measured in the serum collected from NCK56 mono-colonized mice (n = 5, 2 males, 3 females, 17–18 weeks old) and ΔprtX mono-colonized mice (n = 5, 2 males, 3 females, 17–18 weeks old). Serum from a single germ-free control was used to measure background fluorescence. Using a standard of known concentrations of FITC-dextran in PBS, the concentration of FITC-dextran was calculated.

Male C57BL/6 mice (6–8 weeks old) were purchased from Charles River (Sherbrook, Canada) and maintained in the animal care facility at the University of Manitoba under specific pathogen-free conditions. Mice were anaesthetized using Isoflurane (Abbott, Toronto, Canada). PE-90 tubing (10 cm long; ClayAdam, Parisppany, NJ) that was attached to a tuberculin syringe (BD, Mississauga, Canada) was inserted 3.5 cm into the colon and colitis was induced by intra-rectal administration of 100 μl of 4 mg of DNBS solution (ICN Biomedical Inc. Aurora, OH) in 30% ethanol (Sigma, Mississauga, Canada) and left for 3 days (DNBS + ethanol 30%, n = 6)6 31 (link). Controls were time matched and consisted of mice that received intra-rectal administration of 100 μl of 1% phosphate buffer saline (PBS 1%, n = 6) or 100 μl of 4 mg of DNBS solution in 1% PBS (DNBS + PBS 1%, n = 6) or 100 μl 30% ethanol (ethanol 30%, n = 6).