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LAB 2 LIGAND-MEDIATED CALCIUM SIGNALING IN ANIMAL CELLS
I. Introduction
Free cytoplasmic calcium is a major intracellular signal or “second messenger” which regulates a variety of processes in many different cell types. In hormone-secreting cells a rise in calcium is often the signal that triggers exocytosis and a variety of other events. A new class of dye molecules has been developed that allows for the visualization of free cytoplasmic calcium. These are dye molecules that fluoresce only when bound to calcium, this process of the release of free cytoplasmic calcium is visualized utilizing a fluorescence microscope.
A number of the calcium signaling pathways rely on membrane associated lipids. The classic example of a membrane lipid-based signaling pathway is one that uses the membrane-associated enzyme called Phospholipase-C (PLC). PLC is a family of enzyme systems in animals and fungi that cleave the membrane a membrane phospholipid called PIP2 into two subunits DAG and IP3.
PLC’s can be activated a number of different ways, one is by the activation of a G-protein when agonist (Vasopressin, Angiotensin II) binds to the membrane receptor. For today’s lab we are interested in the production of IP3 which binds to a ligand-gated channel in the endoplasmic reticulum where calcium ions are stored. This causes the release of calcium ions into the cytoplasm, which in turn activate a variety of cellular processes.
Fluorescence illumination and observation is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences. Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime. There are a number of commercially-available fluorescent indicators, we will be using Fluo-4 AM which is a new fluorescent indicator that provides brighter images of intracellular Ca++ dynamics compared to its widely used predecessor Fluo-3 or Fluo-2. When considering any fluorescent dye molecule one looks at the excitation spectra, or the wavelengths necessary to get the dye molecule to fluoresce, and the emission spectra, or the wavelengths that are given off and seen.
You then match these to the available optics (fluorescent “cubes”) in your fluorescent microscope (it’s the green, marked WB cube in our Olympus fluorescent microscope). We will be using the fluorescent microscope setup in my office (may be a little tight fit to get us all in). Below is the schematic of the light path for the Olympus IX 70 inverted fluorescent microscope we will be using for today’s lab.
II. Objectives:
A. Observe animal cells in culture B. Learn basic principles of fluorescent microscopy C. Load animal cells with fluorescent calcium indicator D. Observe (we hope) free calcium release in response to hormones
III. Procedure
The type of animal cells used will depend on the type of cells available at the time of the laboratory. The cell culture technique will vary depending on the cells of interest. The basic format will be to obtain cells growing on coverslips in small culture chambers (see figure below).
The cells will then be loaded with Fluo-4 AM using the following procedure.
1. Remove media from cells with pasture pipette or 5 ml pipette
2. Rinse cells twice with 3ml (each time) 37C PBS buffer solution very carefully. If media is present in the cultures, it tends to mess up the fluorescence of the cells. We want to keep the cells at 37C as much as possible and avoid washing them off the bottom of the slides.
3. Add 3 ml of the high or low dye concentration in PBS to the rinsed cells and incubate for 20 (15-45 minutes will work) min at 37C
4. Carefully remove dye solution and carefully add 3 ml of 37C PBS and incubate cells for another 20 minutes at 37C. Longer than 45 minutes and cells do not light up. This gives the cells a chance to recover and gives the cytoplasmic esterases a chance to act on the dye.
5. Place the cells on the fluorescent microscope, get in focus and start a timelapse series (see instructor). Then add 20 ul of the hormone or control (PBS buffer) and look for fluorescence which would indicate binding of hormone to membrane receptors, activating PLC, generating IP3 and a release of free cytoplasmic Ca++.
6. Each group of two students should have 2-3 chamber slides. Let’s use the following experimental design which has two replicates per treatment condition. You will add the appropriate solution (at 37C) to your dye-loaded cells and score fluorescence.
Notes:
Do not use any media or buffer with phenyl red, as this quenches fluorescence.
Do not make up the Fluo-4 for loading in media. The seria in the media will inactivate the dye, and the phenyl red will quench fluorescence.
For Fluo-4 AM loading, use of the minimum concentration of AM ester necessary to get an adequate signal (usually as low as 1 μm and rarely above 5 μm) is recommended to reduce artifacts due to incomplete enzymatic hydrolysis of the AM esters.
Higher AM ester concentration must be avoided due to the necessity for more extensive intracellular esterase activity and to avoid the accumulation of both the lipophilic AM esters in membrane lipids and to formaldehyde from AM ester hydrolysis.
With most cells, loading requires 15-45 minutes at 37°. Ours worked well with 20 minute incubation (. Use of excess dye to try to force loading is usually not advisable due to difficulty in washing out unhydrolyzed AM esters.
Stock solutions of 10 or 20 mM of dye/Pluronic/DMSO can be made up dehydrated, aliquoted, kept dessicated at -20° and protected from light. Working solutions of 50 or 100 uM are prepared in the aqueous buffer on the day of the experiment. These solutions may be cloudy since at higher ionic strengths, the ester is relatively insoluble and tends to form macroscopic clumps. Some of these gross precipitates can be dispersed by sonication.
IV. Questions/Data Collection:
1. What is the purpose of the Acetoxymethyl (AM) form of the fluorescent dye?
2. What is the purpose of Pluronic/DMSO solution?
3. Why do we have to match the absorption and emission spectra of a given dye molecule to the optics of our microscope?
4. Why do we add the dye to cells in serum-free media or buffer solution?
5. What are the steps in the pathway that would allow for the release of calcium with the addition of a hormone like insulin?
6. Explain the results of your experiment (why did it work/why did it not work/what does it mean)
V. Materials:
1. Animal cell line with media, antibiotics, and buffer solutions for that line
2. Lab-Tek chamber coverslip slide chambers
3. Fluo-4 AM dye comes in 50 ug ampules (Molecular Probes F-14201). Fluo-4 keeps for a year or less when stored at -20C.
4. Pluronic F-127 in 20% DMSO solution (molecular probes P-3000MP) Optional
5. 37C incubator, Sterile 1X PBS solution
6. Eppendorff pippetors and tips
7. 1, 5, and 10 ml pipettes and pippetors
8. Fluorescent microscope and camera with appropriate filter cubes
9. Vasopressin is made up in 100 UI/ml concentration, (Sigma V0377 – 100 IU)
10 Angiotensin II (Sigma A9525 5X1mg) make up in 1 ml of 1mg/ml and 1 ml of 0.1 mg/ml
11. DMSO ampoules 5 X 5ml Sigma 44K2303
12. Make up dye solution immediately before lab
a. Fluo-4 AM dye comes in 50 ug ampules (Molecular Probes F-14201).
b. Solubilize contents of one 50 ug ampule with 10-25 ul DMSO, the less DMSO, the better the cells like it.
c. To get different concentrations of dye in PBS use the following dilutions:
Hi Conc. All Dye in solvent (10-25 ul) to 25 ml of 37 C PBS this gives 2 ug/ml
Lo Conc. All Dye in solvent (10-25 ul) to 12.5 ml of 37C PBS, this gives 4 ug/ml.
Use less than 0.1% of DMSO in the loading solution added to cells
VI. References
Tsien, R. Y.: A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290:527-528, 1981.
Tsien, R. Y., Pozzan, T., and Rink, T. J.: Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J. Cell Biol. 94:325-334, 1982.
Hill, R. S., Oberwetter, J. M. and Byd, A. E. III: An increase in cAMP levels in a beta cell line potentiates insulin secretion without altering the cytosolic free calcium concentration. Diabetes, In Press, 1987.
Rajan, A. S., Hill, R. S. and Boyd, A. E. III: Adenosine 3', 5'-cyclic monophosphate (cAMP) raises cytosolic calcium ([Ca2+]i ) in beta cells. ASCI Abstract, Clin. Res. 35:515A, 1987.
Boyd, A. E. III, Hill, R. S., Oberwetter, J. M., and Berg, M.: Calcium dependency and free calcium concentrations during insulin secretion in a hamster beta cell line (HIT cells). J. Clin. Invest. 77:774-781, 1986.
Wollheim, C. B. and Biden, T. J.: Second messenger function of inositol 1,4,5-trisphosphate: early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells. J. Biol. Chem. 261:8314-8319, 1986.
Tsien, R. Y., Rink, T. J. and Poenic, M.: Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy. Cell Calcium. 6:145-157, 1985.
Huyghes, M. R., Schrader, TS, O’Malley, B. W. 1990. Laboratory methods Manual for hormone action and molecular endocrinology. Department of Cell Biology, Baylor College of Medicine,Texas Medical Center, Huston, TX.
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