5-Nitro BAPTA

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
5-Nitro BAPTA, designed to a red fluorescent probe for cytoplasmic Ca²⁺ with strong emission in the long-wavelength region^[1].
General procedure for fluorescence imaging of cultured Hela cells^[1]:
1.Plate cells onto a 35-mm poly-L-lysinecoated glass-bottomed dish (Matsunami) in DMEM supplemented with 10% (v/v) fetal bovine serum, 1% penicillin and 1% streptomycin.
2. Remove DMEM, wash the dish with HBSS 3 times, and then add CaTM-2 AM (3 μM) in Hanks’ Balanced Salt Solution (HBSS) containing 0.3% DMSO as a cosolvent.
3. Incubate at 37°C for 30 min, remove medium and wash dishes with HBSS 3 times. The cells can be observed in HBSS.
4. Capture fluorescence images with excitation and emission wavelength of 590/610-680 nm.
General procedure for fluorescence imaging of slices^[1]:
1. Incubate slide cultures with 2 mL dye solution at 37 oC for 40 min. The dye solution is artificial cerebrospinal fluid (aCSF) containing 10 μM CaTM-2 AM, 0.01% Pluronic F-127, and 0.005% Cremophor EL. aCSF consisted of : 126 mM NaCl, 26 mM NaHCO₃, 3.5 mM KCl, 1.24 mM NaH₂PO₄, 1.3 mM MgSO₄, 1.2 mM CaCl₂, and 10 glucose.
2. Wash slieds with aCSF three times and recover in 2 mL aCSF at 37 oC for 45 min, during which 2 μL of 1 mM Acridine orange was added to the aCSF at time 40 min.
3. Transferre slice cultures into a recording chamber heated at 35 oC and continuously perfused with aCSF at 2 mL/min.
4. Acqure images at 10 frames/s with a Nipkowdisk confocal unit (CSUX-1, Yokogawa Electric, Tokyo, Japan), cooled CCD camera (iXon DU897, Andor, Belfast, UK), a water-immersion objective lens (16×, 0. NA, Nikon, Tokyo, Japan), and image acquisition software (Solis, Andor Technology, Belfast, UK).
5. Set the excitation wavelength to 488 nm (7 mW) and 568 nm (15 mW) for Acridine orange and CaTM-2 with an argon-krypton laser (641-YB-A01; Melles Griot, Carlsbad, CA, USA) and set the emission wavelength to 520-535 nm and 617-673 nm band-pass emission filters, respectively.
6. Analysis data with custom-made software written in Microsoft Visual Basic.
7. Calculate fluorescence change ΔF/F as (Ft-F0)/F0, where Ft is the fluorescence intensity at frame time t, and F0 is the average baseline.

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