Materials and Reagents

15 ml centrifuge tube (VWR, catalog number: 89004-368 )
Clear flat-bottom black 96-well culture trays with lid (Corning, catalog number: 3904 )
Wild type (WT) human 1321N1 astrocytoma cells devoid of P2 receptors and hHA-P2Y₂R expressing 1321N1 cells (gift from Dr. Gary A. Weisman, University of Missouri)
DPBS
Trypsin (TrypLE Express) (Thermo Fisher Scientific, Gibco^TM, catalog number: 12604013 )
Probenecid (water soluble) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P36400 )
Adenosine 5’-triphosphate disodium salt (ATP) (Tocris Bioscience, catalog number: 3245 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: C1016 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
HEPES (Sigma-Aldrich, catalog number: H0887 )
Glucose (Sigma-Aldrich, catalog number: G8270 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
Fura-2-acetoxymethyl ester (fura-2 AM) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: F1225 )
Pluronic F-127, 20% (w/v) stock in DMSO (Thermo Fisher Scientific, Invitrogen^TM, catalog number: P3000MP )
Dulbecco’s modified Eagle’s medium–low glucose (DMEM) (Sigma-Aldrich, catalog number: D2902 )
Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
Antibiotic/antimicotic (Sigma-Aldrich, catalog number: A5955 )
Geneticin, G418 (Thermo Fisher Scientific, Gibco^TM, catalog number: 10131035 )
HEPES buffered saline (HBS), pH 7.4 (see Recipes)
Dye solution (see Recipes)
Medium for wild type (WT) human 1321N1 astrocytoma cells (see Recipes)
Medium for hHA-P2Y₂R expressing 1321N1 cells (see Recipes)
Low serum medium (see Recipes)

Equipment

Tecan Infinite M200^® (Tecan Trading, model: Infinite^® M200 )
Fisher Scientific Mini vortex (Fisher Scientific)
Eppendorf 5810R Benchtop centrifuge (Eppendorf, model: 5810 R )
Biorad TC10 cell counter^® (Bio-Rad Laboratories, model: TC10^TM Automated Cell Counter )
Note: This product has been discontinued.
Fisher Scientific Isotemp CO₂ Incubator (5% CO₂, 37 °C) (Fisher Scientific)
Laminar flow biological hood

Software

i-Control^® (Tecan Trading AG, Switzerland)
Excel^® (Microsoft)
GraphPad Prism^® (GraphPad Software Inc., La Jolla, CA)

Procedure

Cell culture and fura-2 loading
1. Maintain cells with the appropriate media in an incubator (37 °C, 5% CO₂).
2. Grow cell to a 90% confluency, discard the cell medium, wash with DPBS and detach with trypsin.
3. Re-suspend detached cells with 5 ml of cell culture medium and transfer to a 15 ml centrifuge tube.
4. Centrifuge at 300 x g for 5 min.
5. Discard cell culture medium and re-suspend cells with 5 ml of low serum medium.
6. Seed cells 16 h before experiments in clear flat-bottom black 96-well culture trays at a density of 3.0 x 10⁴ cells/well (80-90% confluent).
7. Discard the medium by carefully inverting the plate and wash once with 200 μl HEPES-buffered saline. Carefully discard HBS by inverting the plate, replace with dye solution and incubate cells 1 h at RT in the dark.
8. Remove the dye solution by inverting the plate, wash twice with HBS and cover with 200 μl of HBS supplemented with 2.5 mM probenecid for at least 20 min at RT to allow for fura-2 AM de-esterification.
Note: Cell medium must be without phenol red to avoid fluorescence measurement interference. To avoid evaporation remember to put plate lid back during incubations.
Instrument setup
1. Open the Tecan i-Control software and enter the following parameters:
  
  Table 1. Dual wavelength Ca²⁺ indicator assay measurement parameters and instrument settings for the Tecan Infinite M200^® with injector. Optimized settings to measure ATP stimulated intracellular Ca²⁺ mobilization in 1321N1 astrocytoma cells.
2. Before performing the assay, it is recommended to clean the injectors and tubing first with distilled water, then with HBS.
3. Prime injector A with 1.1 mM ATP solution.
4. Set the measurement parameters, insert the plate, remove the plate lid and start the run. Determine fluorescence emission intensity at 510 nm in individual wells alternating excitation wavelengths of 340 and 380 nm every 3 sec for 40 acquisition cycles, using the parameters specified in Table 1.

Data analysis

To express in intracellular calcium concentration in cells, calculate the ratio of 510 nm emission, in response to 340/380 nm excitation.
Normalize Ca²⁺ mobilization using the average of the first 9 cycles (unstimulated) as baseline.
See Appendix for data processing example.

Notes

Fura-2 AM, under certain staining conditions can accumulate into cell organelles (endoplasmic reticulum, endosome/lysosome, mitochondrion, nucleus). Combinations of dye concentration, incubation time and temperature must be tested to minimize the amount of dye that compartmentalized into organelles (tested by confocal microscopy). Experimental conditions used in this protocol have been optimized for 1321N1 astrocytoma, C6 Glioma and DI TNC cell lines.
Optimal fura-2 loading time and de-esterification time may be determined empirically depending on the cell type. Fura-2 AM, Pluronic F-127, Probenecid concentrations and incubation time stated in this protocol have been optimized for 1321N1 astrocytoma, C6 Glioma and DI TNC cell lines.
Receptor agonist solution preparation:

Agonist solution needs to be prepared taking in consideration well volume and the volume to be dispensed by the instrument injectors.
E.g., Well initial volume = 200 μl
Volume to be dispense = 20 μl
Well final volume = 220 μl
Desired agonist concentration = 100 μM ATP
Agonist stock concentration = 1.1 mM ATP

Tecan i-Control parameters are optimized for this protocol and must be determined empirically.
HBS can be stored at 4 °C. Allow to reach room temperature before use.
Store Pluronic F-127 20% solution at room temperature. If precipitation is observed, the product may be resolubilized by heating at ~40 °C and vortexing before use.

Recipes

HEPES buffered saline (HBS), pH 7.4
145 mM NaCl
5 mM KCl
1 mM CaCl₂
1 mM MgCl₂
10 mM HEPES
10 mM glucose
Dye solution
HEPES buffered saline (HBS), pH 7.4
0.1% BSA (w/v)
5 μM fura-2 AM
0.05% Pluronic F-127
Medium for wild type (WT) human 1321N1 astrocytoma cells
DMEM
10% fetal bovine serum (FBS)
1% antibiotic/antimicotic
Medium for hHA-P2Y₂R expressing 1321N1 cells
DMEM
10% fetal bovine serum (FBS)
1% antibiotic/antimicotic
500 μg/ml Geneticin, G418
Low serum medium
DMEM
1% fetal bovine serum (FBS)

Acknowledgments

Partial support to MMA and NAM was received from the Associate Deanship of Biomedical Sciences of the UPR-MSC. Experiments were performed at the Molecular Sciences Research Center of the University of Puerto Rico. 1321N1 astrocytoma cells (WT and P2Y₂R expressing) were a gift from Dr. Gary A. Weisman, University of Missouri.

References

Burgos, M., Neary, J. T. and Gonzalez, F. A. (2007). P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells. J Neurochem 103(5): 1785-1800.
Martínez, N. A., Ayala, A. M., Martínez, M., Martínez-Rivera, F. J., Miranda, J. D. and Silva, W. I. (2016). Caveolin-1 regulates the P2Y2 receptor signaling in human 1321N1 astrocytoma cells. J Biol Chem 291(23): 12208-12222.