HMBI

Guide (The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

1. Stock Solution Preparation
1.1 Solvent: DMSO^[1].
1.2 Concentration recommendation: 10 μM^[1]; alternatively, 2% (20 mg HMBi per fermentation system)^[2].

2. Working Solution Preparation
2.1 Diluent: Britton-Robinson (BR) buffer^[1].
2.2 Working concentration: 5 μM^[1].
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 For live adherent cells (SiHa, HeLa cells)^[1]:
3.1.1 Sample handling: No trypsinization specified for live cell staining.
3.1.2 Incubation conditions: Incubate with 5 μM HMBI for 30 min.
3.1.3 Washing steps: Not specified.
3.2 For rumen fluid from rumen-cannulated Hu sheep^[2]:
3.2.1 Incubation conditions: Incubate fermentation systems containing 2% HMBi at 39°C for 3 h, 6 h, 9 h, 12 h, or 24 h under anaerobic conditions.
3.2.2 Washing steps: Not specified.

4. Controls
4.1 Negative controls: DNase-treated fixed cells, CCCP-treated live cells, H₂O₂-treated live cells, paraformaldehyde-fixed cells^[1]; fermentation systems with no HMBi added^[2].
4.2 Positive controls: Live cells stained with commercial nucleus probe Hoechst 33342 and mitochondrial probe MitoTracker Deep Red FM (MTDR)^[1].
4.3 Blank control: Excludes reagent fluorescence interference^[1].

5. Detection & Analysis
5.1 Instrument type:
5.1.1 Confocal laser scanning microscopy^[1].
5.1.2 Gas meter, acidity meter, UV-Vis spectrophotometer, gas chromatography, automatic amino acid analyzer, high-throughput sequencing platform^[2].
5.2 Ex/Em wavelengths:
5.2.1 Green channel: Ex=405 nm, Em=510-560 nm^[1].
5.2.2 Red channel: Ex=543 nm, Em=560-660 nm^[1].
5.3 Result analysis:
5.3.1 For live adherent cells: Green fluorescence localizes to nuclei, red fluorescence localizes to mitochondria; fluorescence color change correlates with pH (green emission in acidic environment (nuclei), red emission in basic environment (mitochondria))^[1].
5.3.2 For damaged/apoptotic adherent cells: Red fluorescence intensity in mitochondria dramatically decreases, while green fluorescence intensity in nuclei remains almost unchanged; shrinking and pyknotic nuclei are observable^[1].
5.3.3 For rumen fluid fermentation systems: Cumulative gas production is higher in the HMBi group than the control group at 3 h; total volatile fatty acids (VFAs), microbial protein (MCP) concentration, acetate, and acetate to propionate ratio are higher in the HMBi group than the control group; NH₃-N concentration is lower in the HMBi group than the control group; dry matter digestibility at 12 h and 24 h is higher in the HMBi group than the control group; methionine concentration in rumen fluid is higher in the HMBi group than the control group; relative abundance of Bacteroidetes, Firmicutes, and Synergistetes is higher in the HMBi group than the control group; relative abundance of Proteobacteria is lower in the HMBi group than the control group; relative abundance of Prevotella_1 is higher in the HMBi group than the control group; relative abundance of Ruminobacter is lower in the HMBi group than the control group^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.