- Neural stem cells, cultured on poly-D-lysine coated 96-well plate or other culture vessel
- Hanks’ Balanced Salt Solution (HBSS) (Cat. no. 14025-134)
- Fluo-4, AM (Cat. no. F14201)
- Pluronic® F-127 (Cat. no. P-3000MP)
- DMSO (Dimethylsulphoxide) (Sigma, Cat. no. D2650)
- Neurotransmitters or ligands (e.g., acetylcholine, glutamate)
Tools and Equipment
- Inverted microscope (e.g., Nikon T2000)
- Illumination system (e.g., Sutter Instruments Lambda DG-4)
- Digital camera (e.g., Hamamatsu ORCA-ER)
Fluo-4 AM loading solution consists of 3 μM fluo-4 AM (reconstituted in DMSO) and 0.1% Pluronic® F-127 in Hanks’ Balanced Salt Solution (HBSS). Use the fluo-4 AM loading solution as soon as possible after preparation to avoid decomposition with subsequent loss of cell loading capacity.
- To reconstitute fluo-4 AM, add 44 μL of DMSO to one vial of fluo-4 AM (50 μg) and vortex thoroughly. You may store the fluo-4 AM reconstituted in DMSO protected from light, frozen, and desiccated for up to one week.
- Add 9 μL of Pluronic® F-127 to the reconstituted fluo-4 AM and vortex thoroughly. Note: Because fluo-4 AM is relatively insoluble in aqueous solutions, addition of the low-toxicity dispersing agent Pluronic® F-127 facilitates cell loading. However, Pluronic® F‑127 may decrease the stability of AM esters, so it should only be added to working stocks (i.e., the loading solution).
- Add 50 μL of the ~860 μM fluo-4 AM/ Pluronic® F‑127 solution to 14.3 mL of HBSS.
Loading NSCs with Fluo-4 AM Loading Solution
- Wash the NSCs with 100 μL of Hanks’ Balanced Salt Solution (HBSS).
- Load the NSCs with 100 μL of fluo-4 AM loading solution per well of a 96-well plate. You may adjust the volume as appropriate to other culture vessels.
- Incubate the NSCs in the dark at room temperature for ~60 minutes.
- Wash the fluo-4-loaded NSCs with 100 μL of HBSS and maintain at room temperature in the dark until data acquisition
- Place the 96-well plate containing the fluo-4-loaded NSCs in an inverted microscope (e.g., Nikon T2000) for visual inspection and fluorescent imaging.
- To acquire and analyze data, define regions of interest around a random series of cells using your software of choice (e.g., MetaFluor, MDS Analytical Technologies). Note: The NSCs should display a typical neuronal morphology with dendritic and axonal processes clearly recognizable by cellular polarity and proportionate size.
- Identify 50–100 neurons for data acquisition and analysis in each well examined.
- Excite the NSCs with 488-nm light (e.g., Lambda DG-4 light source) and collect images from 520-nm emitted light with a CCD or digital camera (e.g., ORCA-ER).
- Challenge the cells in one well with a neurotransmitter or other ligand. For example, add 20 μL of 3 mM acetylcholine to achieve a final concentration of 500 μM acetylcholine in the well.
- Collect the data using the appropriate software (e.g., MetaFluor, MDS Analytical Technologies).
- Repeat the procedure for each neurotransmitter or ligand of interest in separate wells. Use the following final concentrations for each well: 500 μM glutamate, 500 μM dopamine (add 500 μM ascorbic acid with dopamine to prevent dopamine oxidation), 500 μM γ-aminobutyric acid, and 500 μM ATP.
- Integrate the acquired fluo-4 520-nm emission signal for each region of interest, normalize to the first ten data points (F/F0) and then plot against time.
- Set the response criteria. For example, a NSC might be considered responsive to a given neurotransmitter or ligand if the resulting normalized signal rises more than 10% within 60 seconds following neurotransmitter addition compared to the baseline signal. The number of NSCs that exhibit clear changes in intracellular Ca2+ ([Ca2+]i) depends on the neurotransmitter and differentiation state of the NSCs.