Histamine elisa kit

Pre‐treatment to examine the immune‐stimulating effect of the HBP vacuoles was conducted. Histamine released from the stimulation of HBP vacuoles and Lipopolysaccharide (LPS) in RAW 264.7 cells was estimated using Histamine ELISA kit (Enzo life science). The experiment was conducted as described in the product manual. RAW 264.7 cells were adjusted to 5 × 10⁴/ml, cultured for 24 h and starved for 12 h. Cells were pre‐treated with HBP vacuoles for 6 h at various concentration of (1–10) ng/ml, washed twice with DPBS and then proceeded to the next step. Cells were then treated with LPS at 1 μg/ml for 24 h. The HA concentration of the supernatant was estimated using Histamine ELISA kit.
Histamine ELISA was performed to confirm that HA released from LPS stimulation of the RAW 264.7 cells (Dy et al., 1981 (link)) could be inhibited by treatment with HBP vacuoles. RAW 264.7 cells were adjusted to 5 × 10⁴/ml, cultured for 24 h and starved for 12 h. Cells were pre‐treated with LPS at 1 μg/ml for 24 h. The cell supernatant was treated with various concentration of HBP vacuoles of (1–10) ng/ml for 2 h. Then, the HA concentration of the supernatant was estimated by Histamine ELISA. pVMA11 vacuoles were used to compare the effects on the recombinant vacuoles as controls.

Production of histamine from L-histidine by L. reuteri was measured via ELISA (Enzo Life Sciences, Inc., Farmingdale, NY). L. reuteri was grown overnight in MRS as described above, 1 ml aliquots of 2 × 10⁹ CFU were pelleted at 3220 × g for 10 min, washed twice with sterile saline, and resuspended in one of the following conditions: sterile saline, saline with 3% maltose, saline with 2% v/v glycerol, 4 mg/ml L-histidine (Sigma-Aldrich, St. Louis, MO), 4 mg/ml L-histidine with 3% maltose, or 4 mg/ml L-histidine with 2% v/v glycerol. 5 mg of DM containing either 4 mg/ml or 30 mg/ml L-histidine were added to media lacking L-histidine, so that the only source of L-histidine for L. reuteri was as cargo diffusing out of the DMs. Each condition was then incubated at 37°C for 2 h, after which time the contents were pelleted and the supernatant was removed for histamine quantification via a histamine ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY) following the manufacturer's instructions without modifications. All conditions were done in at least triplicate.

Skin punch biopsies (4 mm) were collected from the dorsal skin of mice immediately after euthanasia. Biopsies were incubated in DMEM plus antibiotics with 2 mM L-glutamine and 10 mM HEPES (Thermo Fisher) for 24 h at 37°C in a 5% CO₂ incubator. The conditioned media was snap-frozen and stored at −80°C until analyzed.
Whole blood was collected by cardiac puncture into EDTA tubes (T-MQK, Terumo Medical Corp., Somerset, NJ, USA). Plasma was separated by centrifugation of whole blood at 1,200 g and 4°C for 10 min. Equal volumes of plasma and methanol were added to a 1.5 ml microcentrifuge tube, mixed, and incubated on ice for 3 min. Supernatant was collected after the mixture was centrifuged at 5,000 g for 5 min. The extract was then dried in a centrifugal concentrator for 2–3 h at room temperature and reconstituted with assay buffer prior to analysis.
The processed sample was analyzed using the Histamine ELISA kit (ENZ-KIT140-0001, Enzo LifeScience). Assay results were collected and calculated using the Synergy HT plate reader and Gen5™ 1.0 data analysis software (BioTek).