TCM-4COOH

Operating Instructions (The following is a recommended experimental protocol for guidance only; adjust according to actual requirements)

1. Stock Solution Preparation
1.1 Solvent: Most dyes are typically dissolved in organic solvents, such as anhydrous DMSO.
1.2 Recommended Concentration: Prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Culture medium; deionized water; ammonia buffer (pH=10).
2.2 Working Concentration: 10 μM-1 mM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedures
3.1 Sample Type Description
3.1.1 Adherent cells (PC12 and HeLa cells, seeded in confocal culture dishes and allowed to adhere for 12 h before imaging)^[1].
3.1.2 Tissue samples (fresh bovine femur, cleaned and surface soft tissues removed; 20×1 mm marks drawn on the bone surface to simulate microcracks)^[1].

3.2 Incubation Conditions
3.2.1 Ionomycin-pretreated adherent cells: Incubate with 10 μM TCM-AM for 1 h in a humidified environment at 37℃ with 5% CO₂.
3.2.2 Bone microfracture samples: Incubate with 1 mM TCM-4COOH for 5 s.
3.2.3 In vitro Ca²⁺ response assay: Incubate with CaCl₂ solution, stir at 750 rpm for 2 min; shake in a 96-well plate for 2 min.
3.2.4 Ion solution detection in 96-well plates: Incubate with 20 μM-2 mM TCM-4COOH for 1-2 min at 25℃.
3.3 Washing Procedures
3.3.1 Adherent cells: After ionomycin incubation, wash 3 times with phosphate-buffered saline (PBS).
3.3.2 Bone microfracture samples: Wash with deionized water for 10 s after staining.

4. Control Setup
4.1 Commercially available calcium probes Fluo-4-AM (HY-101896)/Fluo-3-AM (HY-D0716) are used as controls for in vitro Ca²⁺ response and cellular calcium overload imaging.
4.2 Calcein (HY-D0040) is used as a control for bone microfracture staining.
4.3 Set up an untreated cell group to eliminate autofluorescence interference.
4.4 Set up a blank control group with TCM-4COOH without target ions.

5. Detection and Analysis
5.1 Instrument Types: Laser confocal scanning microscope; multi-mode microplate reader; UV lamp; fluorescence microscope; two-photon microscope.
5.2 Excitation/Emission Wavelengths:
5.2.1 TCM-AM: Ex=476 nm, Em=550-650 nm.
5.2.2 TCM-4COOH: Ex=477 nm, Em=600 nm; Ex=477 nm, Em=603 nm; Ex=478 nm, Em=600 nm.
5.2.3 Bone microfracture imaging: Ex=365 nm.
5.3 Result Analysis
5.3.1 Fluorescence intensity changes: When the Ca²⁺ concentration ranges from 0 to 5.0 mM, the fluorescence intensity increases by up to 21-fold; the fluorescence intensity of calcium-overloaded PC12 cells stained with TCM-AM is 4.8 times that of normal cells, while that of HeLa cells is 7.5 times; the fluorescence intensity of bone microcracks stained with TCM-4COOH remains stable even after 12 h of immersion; the fluorescence intensity increases significantly after incubation with Ca²⁺ and Mg²⁺, with a stronger intensity response to Ca²⁺ than to Mg²⁺; the fluorescence intensity increases significantly upon binding to Ca²⁺.
5.3.2 Fluorescence localization: The fluorescence generated by TCM-AM is mainly localized in the endoplasmic reticulum of calcium-overloaded cells; fluorescence localizes to regions with high Ca²⁺ concentrations.
5.3.3 Fluorescence properties: TCM-4COOH exhibits bright red/orange fluorescence after binding to high-concentration Ca²⁺; TCM-4COOH produces fluorescence after binding to Ca²⁺ via its carboxyl groups, and the mechanism of fluorescence enhancement is restricted intramolecular motion.