The following protocol describes how to perform fluo-4-based measurements of cytosolic calcium changes in neural stem cells in response to neurotransmitter applications.

Required Materials


  • 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 (dimethyl sulfoxide) (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)

Preparing Reagents

Fluo-4 AM Loading Solution

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.

  1. 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.
  2. 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).
  3. 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

  1. Wash the NSCs with 100 μL of Hanks’ Balanced Salt Solution (HBSS).
  2. 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.
  3. Incubate the NSCs in the dark at room temperature for ~60 minutes.
  4. Wash the fluo-4-loaded NSCs with 100 μL of HBSS and maintain at room temperature in the dark until data acquisition

Data Acquisition

  1. 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.
  2. 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.
  3. Identify 50–100 neurons for data acquisition and analysis in each well examined.
  4. 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).
  5. 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.
  6. Collect the data using the appropriate software (e.g., MetaFluor, MDS Analytical Technologies).
  7. 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.

Data Analysis

  1. 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.
  2. 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.
LT155                   17-Mar-2011