Measurement of intracellular calcium of submandibular glands using a high throughput plate reader
Key events in salivary gland calcium signaling
Fura-2 as a calcium sensing dye
High throughput assay of calcium signaling
- HEPES (Sigma-Aldrich, Cat. # H3375)
- NaCl (Sigma-Aldrich, Cat. # S7653)
- KCl (BDH, AnalaR, Cat. # 101984L)
- MgCl2 (Sigma-Aldrich, Cat. # M8266)
- CaCl2 (Sigma-Aldrich, Cat. # C-4901)
- Glucose (Sigma-Aldrich, Cat. # G8270)
- Glutamine (Sigma-Aldrich, Cat. # G7513)
- MEM Non-Essential Amino Acids Solution (100X) (Thermo Fisher Scientific (Life Technologies) Cat. # 11140050)
- Bovine serum albumin (BSA) (Sigma-Aldrich, Cat. # A2153)
- Collagenase from Clostridium histolyticum (Type-4 collagenase) (Sigma-Aldrich, Cat. # C5138)
- Soybean Trypsin Inhibitor (Thermo Fisher Scientific (Life Technologies), Cat. # 17075029)
- Corning® Cell-Tak (Fisher Scientific Ltd, Cat. # 354240)
- NaHCO3 (Sigma-Aldrich, Cat. # S6014)
- Fura-2 AM (Molecular Probes™, Cat. # F-1201)
- Probenecid (Sigma-Aldrich, Cat. # P8761)
- Half-area, 96-well plates ((Fisher Scientific Ltd, Cat. # 10717804)
- Carbachol (CCh) (Santa Cruz Biotechnology, Cat. # sc-202092)
- Ionomycin (IM) (Santa Cruz Biotechnology, Cat. # sc-3592)
- Culture grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Cat. # 276855)
- Dulbecco phosphate buffered saline (DPBS) (Sigma-Aldrich, Cat. # D8662)
- HEPES incubation buffer: 20 mM HEPES, 95 mM NaCl, 4.7 mM KCl, 0.6 mM MgCl2, 1.3 mM CaCl2, 10 mM glucose, 2 mM glutamine, and 1 × minimum Eagle’s medium non-essential amino acids, pH 7.4. The buffer was oxygenated for 20 min before use
- BSA incubation buffer: BSA 1% w/v final added to 25 ml of the HEPES buffer
- Collagenase digestion buffer (CDB): 1.1 mg/ml type-4 collagenase and 1 mg/ml soybean trypsin inhibitor added to 6 ml of BSA incubation buffer
- Sodium bicarbonate (NaHCO3) neutral buffer solution: 0.1 M sodium bicarbonate, pH 8.0 was prepared by dissolving 420 mg NaHCO3 in 50 ml ultrapure distilled water
- For coating of a half area 96-well plate: Dilute 30 µl of Corning® Cell-Tak in 2 ml of the neutral bicarbonate buffer
- Fura-2 AM stock solution: Suspend 1 mg of lyophilized Fura-2 AM with DMSO to yield a 1 mM stock. Aliquot this stock and keep at all times in the dark at −20°C
- 1 M probenecid: Dissolve in 1 M NaOH (50 mg/ml), yielding a clear, colorless solution
- Fura-2 working solution: 4 µl Fura-2 AM stock, 4 µl probenecid 1 M and 4 ml HEPES buffer
1.Assay plate preparation
1.1.On the day preceding the experiment, filter-sterilize the NaHCO3 neutral buffer solution.
1.2.The amount of Corning® Cell-Tak required for each well in the assay plate is calculated according to the manufacturer’s recommendations: 0.56 µg Corning® Cell-Tak/well.
1.3.Dilute the correct amount of Corning® Cell-Tak into the neutral buffer, mix thoroughly, and dispense into the assay plate wells within 10 min.
1.4.Place the cover and incubate the coated assay plate overnight at room temperature.
1.5.On the next day (the day of the experiment), pour the unevaporated Cell-Tak off and wash each well with 200 µl filter-sterile distilled water to remove the bicarbonate.
2.Isolation and preparation of the SMGs
2.1.Dissect the submandibular glands (SMGs) and rinse it with Hanks balanced salt solution.
2.2.Mince the excised SMG with scalpels or curved scissors in a labeled weighing boat, containing 1 ml of the CDB.
2.3.Transfer the gland homogenate to a 50 ml falcon tube and incubate in 4 ml CDB, in a 37°C water bath for 30 min.
2.4.After the digestion is complete, carefully pipette-out the CDB and replace with 6 ml of BSA incubation buffer.
2.5.Shake the tube vigorously by hand for 10 s, in order to disperse the cells into smaller acinar units (Fig. 3).
2.6.Allow the acinar units to settle, then discard the supernatant and replace with HEPES buffer-containing Fura-2 AM.
3.1.To prevent leakage of the dye from the cells, an organic anion transport blocker probenecid was added to the dye buffer to achieve a final concentration of 1 mM.
3.2.Incubate the acinar units (in the falcon tube) in 4 ml Fura-2 working solution in a CO2 incubator at 37°C for 1 h.
3.3.During this period, switch on the FlexStation and adjust the temperature to 37°C.
3.4.After one hour, wash the acinar units with HEPES buffer once.
3.5.Calculate the final HEPES buffer volume to be dispensed on the acinar units according to number of wells to be seeded, using the following formula: final HEPES buffer per gland = number of wells to be seeded × 75 (final volume/well in the assay plate).
3.6.After seeding the HEPES buffer/acinar units into the assay plate, cover it and place it into its allocated position in the FlexStation, until preparation of the compound plate is complete.
4.Compound plate preparation
4.1.Prepare the stock solutions of the cholinergic receptor agonist; carbachol (CCh) and the calcium ionophore; ionomycin (IM) in DMSO.
4.2.Prepare the intermediate and working solutions in DPBS (Table 1).
5.1.Set-up the FlexStation 3 to record changes in calcium signals before (baseline) and after compound additions, as demonstrated in Table 2.
5.2.For baseline fluorescence reading, adjust the settings similar to that demonstrated in Table 2, except that: (1) select an endpoint read type; (2) do not select the compound transfer option.
5.3.Record emission ratios with excitation wavelengths of 340 and 380 nm every 6 s after compound applications, for 3 min.
5.4.Experimental data is processed directly using the SoftMax Pro software (otherwise it can be copied and pasted into any spreadsheet program, such as Microsoft Excel).
|Intermediate||Final conc. (µM) in assay plate||Initial conc. (µM) in compound plate||CCh (µl)||IM (µl)||Buffer (µl)|
|Carbachol stock solution (100 mM): 100 mg CCh in 5.47 ml DMSO||1:100 in DPBS||50||200||40||160|
|Ionomycin stock solution (3 mM): 5 mg IM in 2.23 ml DMSO||No intermediate is required||6||30||2||198|
||Excitation: Lm1 340 nm|
|Lm2 380 nm|
|Emission: Lm1 510|
||96-well Corning half area flat clear bottom|
||Select all (read the whole plate)|
||PMT gain: Medium|
|Flashes per Read: 6|
||Total run time: 3 mins|
|Interval: 6 secs|
|Number of reads: 21|
||Compound 1 (CCh): 75|
|Compound 2 (IM): 100|
||Compound 1 (CCh): 90|
|Compound 2 (IM): 100|
||Compound 1 (CCh): 25|
|Compound 2 (IM): 25|
||Compound 1 (CCh): 2|
|Compound 2 (IM): 2|
||Compound 1 (CCh): 20|
|Compound 2 (IM): 120|
|Compound plate type||Costar 96 opaque 3 ml|
|Pipette tips and layout||Subject to experimental condition|
|Compound and tips column||Subject to experimental condition|
|No trituration (otherwise dislodgment of the Cell-Tak adherent cells will occur)|
|Assay plate preparation||
|Isolation and preparation of the SMGs||
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