Iozzo Renato V. Proteoglycan Protocols

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438 Patel and Iozzo

changes are difficult to distinguish from actual changes in [Ca

] when using

intensiometric indicators. For example, in many cells, dye can be actively extruded

from the cytosol or undergo photobleaching (see Note 1), which results in a decrease

in fluorescence intensity. This decrease in fluorescence would appear as a decrease in

[Ca

] when measured at a single wavelength. For ratiometric dyes such as fura-2,

however, nonreciprocal changes in the fluorescence intensities are cancelled out after

calculation of the fluorescence ratio (Fig. 2B). Ratiometric recording is also less prone

to artefacts arising from cell movement, uneven dye distribution, or inhomogeneities

in cell thickness. Fura-2, then is the dye of choice for single cell Ca

imaging.

1.2. Loading Fluorescent Ca

Indicators Into Cells

Most Ca

indicators are available as acetoxymethyl (AM) esters, rendering them

cell permeable (15). Once inside the cell, endogenous esterases cleave the ester bonds,

releasing the negatively charged dye and essentially trapping it within the cell. Thus,

simple incubation of the cells in a physiological medium supplemented with the AM

Fig. 2. (A) Excitation spectra of fura-2 in the presence of zero and saturating Ca

. The

graph shows fluorescence (F) of the dye measured at 510 nm at a range of excitation wave-

lengths. Binding of Ca

reduces the excitation peak from 360 to 340 nm. Fluorescence of the

dye therefore increases at 340 nm and decreases at 380 nm in response to Ca

. No change in

fluorescence occurs at 360 nm. (B) Ratio measurements cancel out nonreciprocal changes in

fura-2 fluorescence. A schematic fura-2 timecourse recording showing a change in [Ca

] manifest

as a transient increase and decrease in fluorescence at excitation wavelengths of 340 and 380

nm, respectively (left). The response is superimposed on a progressive slow decrease in fluo-

rescence at both wavelengths. These non-reciprocal (Ca

-independent) changes in fluores-

cence are eliminated after calculation of the fluorescence ratio (340/380, right).

Principles of Ca

Imaging 439

ester for a defined period of time results in significant accumulation of the dye within

the cell (see Subheading 3.1.). The ease by which these dyes can be introduced into

cells is a major advantage of using these dyes for monitoring [Ca

1.3. Instrumentation

Central to an imaging system (Fig. 3) is the epifluorescence microscope that houses

appropriate optics to allow delivery of the exciting light and isolation of the emitted

fluorescence. Briefly, cells grown on a glass cover slip are loaded with a fluorescent

indicator and placed into a holder on the stage of the microscope. Excitation light of

the appropriate wavelength for the indicator is selected (usually with a filter) and

directed to the cells via the objective lens through which the cells are imaged. Part of

the resulting emitted fluorescence is collected by the objective and discriminated from

the excitation light by a dichroic mirror. The light is then focused onto a camera, digi-

Fig. 3. Schematic representation of a typical imaging system. Light from an arc lamp

(1) is attenuated by a neutral density filter (2) and passed through a shutter (3) to a filter

wheel (4) that selects the appropriate excitation wavelength. The excitation light (Ex) then

enters the microscope and is reflected by a dichroic mirror (5) up into the objective (6) to

the sample (7) loaded with the fluorescent dye. The emitted fluorescence (Em) is then

collected by the objective and directed back to the dichroic mirror, where it is transmitted

to a second mirror (8) that reflects the light to the camera (9). The shutter, filter wheel, and

camera are computer-controlled.

440 Patel and Iozzo

tized, and stored to a computer. By periodically illuminating the cells through the use

of a computer-controlled shutter, a series of images is collected. Regions of interest,

corresponding to individual cells (or subcellular regions) are selected by the user, and

data are extracted from the image at each time point. Fluorescence intensities/ratios

can then be calibrated to [Ca

] for individual cells in the entire field. A brief description

of the principle components of an imaging system is given in Table 1.

Many imaging systems are now available commercially. Normally, an inverted as

opposed to an upright microscope configuration is adopted, since access to the sample (for

perfusion or microinjection) is unhindered. The choice of camera is one of the most impor-

tant considerations in low-light-level studies. Cameras can be broadly classified according

to their output, which may in the form of a standard video signal (e.g., silicon-intensified

target cameras) that is later digitized or direct digital readout (e.g., cooled charged-coupled-

device cameras). The latter provide better signal-to-noise ratios but are inherently slower.

High-sensitivity cameras allow illumination intensity to be reduced without compromis-

ing signal, thus reducing photobleaching of the dyett. Imaging formats such as 1024 by

1024 pixels are typical. The range of digitisation (8–16 bit) determines the dynamic range

(256–65536 intensity levels per pixel) (see ref. 16 for further details).

1.4 Calibration of Fluorescence Data

Once fluorescence data are acquired, it is relatively straightforward to convert them

to [Ca

]. Data should first be corrected for background fluorescence that is, cell-

independent (“instrument”) noise. For intensiometric indicators, fluorescence inten-

sity (F) is related to [Ca

] by the following equation:

(F – F

min

)·K

max

– F)

where F

min

and F

max

are the fluorescence intensities of the Ca

-free and Ca

-satu-

rated dye, respectively, and K

is the dissociation constant of the dye for Ca

. With

these indicators, an in situ calibration is necessary since measured fluorescence is

dependent on dye concentration (see Subheading 3.4.1.). This involves exposing cells

to a Ca

ionophore such as ionomycin in order to equilibrate Ca

across the plasma

membrane, and then setting the extracellular [Ca

] to zero and saturating levels to

derive F

min

and F

max

, respectively. The parameters are thus determined under similar

dye loading levels as those during experimentation.

For ratiometric indicators, the fluorescence ratio, R (Ca

-bound fluorescence/Ca

free fluorescence) is related to [Ca

] by the following equation:

(R –R

min

· S

max

–R)S

where R

min

and R

max

are the fluorescence ratios of the Ca

-free and Ca

-saturated

dye, respectively, K

is the dissociation constant of the dye for Ca

, and S

is the

ratio of fluorescence values for the Ca

-free and Ca

saturated dye at the wavelength

used to monitor the Ca

-free indicator. For fura-2, R is the fluorescence of the dye at

340 nm excitation/fluorescence of the dye at 380 nm excitation and S

determined at 380 nm. At 37°C, the K

of fura-2 is 224 nM.

[Ca

] =

[Ca

] =

Principles of Ca

Imaging 441

min

and R

max

(from which S

is calculated) can be determined in situ with iono-

phore as with intensiometric indicators. However, as ratios are independent of dye con-

centration, calibration parameters can also be obtained (for the particular instrument

configuration employed) in vitro that is, in the absence of cells (see Fig. 4A; Subhead-

ing 3.3.2.). This is achieved using an intracellular-like solution supplemented with the

free (de-esterified) form of the dye in the presence of BAPTA and excess Ca

to deter-

mine the fluorescence of the Ca

-free and Ca

-bound dye, respectively. Unlike in situ

calibrations, which need to be performed at the end of each experimental run, in vitro

calibrations need only be performed once on the day of experimentation. Furthermore, it

is often difficult to completely equilibrate Ca

across the plasma membrane in in situ

calibrations. However, a major assumption in in vitro calibrations is that the conditions

mimic the environment of the dye within the cell, which may not necessarily be the case.

Fig. 4. In vitro calibration of ratiometric fluorescence data. (A) A cytosol-like calibration

medium (initially Ca

-free) is used to determine calibration parameters (see Table 2). For

fura-2, fluorescence of the medium at 340-nm (solid line) and 380-nm (dotted line) excitation is

acquired first in the absence of dye to determine background fluorescence. Fura-2 free acid

(1 µM) and then CaCl

(500 µM) are added to determine R

min

, R

max

, and S

as shown. These

parameters are used to convert acquired ratio values to [Ca

] using Eq. (2). (B) Autofluorescence

(AF) is measured with either MnCl

or digitonin (see text) and subtracted from all experimental

data prior to calculation of the fluorescence ratio.

442 Patel and Iozzo

442

Table 1

Components of a Single-Cell Imaging System

Component Function Notes

Arc Lamp Provide illumination Xenon arc lamps are preferred over mercury lamps, because they provide a

more even illumination over the range of wavelengths required for excitation

commonly used Ca

indicators.

Neutral-density Filter Attenuate light source Attenuates light evenly at all wavelengths.

Computer-controlled Provide illumination only Prevents premature burning out of filters shutter during acquisition and

shutter unnecessary photobleaching.

Excitation selector Provide appropriate wavelength of Normally achieved with a barrier filter. For dual-excitation studies (as with

light for optimal excitation of the fura-2), it is necessary to have a device for the rapid changing of the excitation

fluorophore wavelength. This can be achieved with a computer-controlled rotating filter

wheel, which houses the appropriate excitation filters (18,19). Alternatively,

a monochromator system can be employed, which is more flexible in that the

number of available wavelengths is essentially unlimited.

Dichroic mirror Reflect excitation light to, Designed to reflect a specific range of wavelengths while allowing light of

and isolate emitted fluorescence other wavelengths to pass through. Chosen such that the cutoff for passing

from sample light is greater than the wavelength of exciting light. Excitation light is

therefore reflected up to the sample, whereas the resulting emitted

light (of longer wavelength) passes through. For fura-2, a dichroic mirror

with a cutoff of 400 nm is appropriate to separate excitation light (340/380 nm)

and fluorescence emission (peak 510 nm).

Objective To deliver excitation light 16 × or 20 × magnification is sufficient to accurately resolve most single

and collect emitted fluorescence cells. Higher magnification (40 × /63 ×) is required for subcellular recording.

Must efficiently transmit light at wavelengths to be used. Fura-2, for example

requires quartz or Fluor objectives in order to pass light of 340 nm.

Lenses with high numerical apertures (NA), an index of light-collecting

capacity, are preferred.

Sample chamber Chamber that fits onto In an inverted microscope configuration, the cover slip itself forms the base

microscope stage and houses the of the chamber. It is usually thermostatted to physiological temperatures.

cover slip containing adherent cells

Principles of Ca

Imaging 443

443

Emission selector Select appropriate emission A filter designed to pass a broad range of wavelengths (long-pass) is

wavelengths normally used, in order to maximize the fluorescence signal, but sufficiently

removed from the cutoff wavelength of the dichroic mirror in order to filter

out stray excitation light. For Fura-2, a 420- to 600-nm filter can be used

with a 400-nm cutoff dichroic mirror.

For dual-emission dyes such as indo-1, a filter system similar in design to

that used to select alternate excitation wavelengths can be fitted to the exit port

of the microscope.

Camera Capture emitted fluorescence Silicon-intensified target (SIT) cameras or cooled charged-coupled-device (CCD)

cameras are commonly used.

444 Patel and Iozzo

Before calculation of fluorescence ratios in dye-loaded cells, it is important to sub-

tract any fluorescence not emanating from Ca

-sensitive dye from the measured sig-

nal. Such autofluorescence can derive from many sources, including flavoproteins and

reduced adenine nucleotides. Autofluorescence is determined by first eliminating the

fluorescence due to the dye and then subtracting the residual fluorescence from the

data. For fura-2, this can be achieved by introducing Mn

into the cell (using iono-

phore), which quenches dye fluorescence (see Fig. 4B, Subheading 3.4.1.). Note that

with intensiometric indicators, autofluorescence is cancelled out in both the numera-

tor and denominator of Eq. (1), and thus does not need to be determined separately.

In some cells, AM loading can result in accumulation of the dye into noncytosolic

compartments such as the endoplasmic reticulum. Since Ca

levels in the endoplas-

mic reticulum are much higher than in the cytosol, compartmentalized dye will, under

normal conditions, remain saturated, thereby contributing to background fluorescence.

In this case, the Mn

quench method for determining autofluorescence is unsuitable,

because ionomycin will equilibrate Mn

into most cellular compartments. An alterna-

tive method to determine autofluorescence in cells where there is significant compart-

mentalization of dye (or when using dyes, such as fluo-3, that are not completely

quenched by Mn

) is to permeabilize selectively using, the plasma membrane with

detergents such as digitonin (see Fig. 4B, Subheading 3.4.2.). This method effects

release of just the cytosolic dye from the cell. More problematic is compartmentaliza-

tion of dye into organelles such as mitochondria, where indicators used for measuring

cytosolic Ca

will also report mitochondrial Ca

changes. In such cases “mixed”

signals will result, since cytosolic and mitochondrial Ca

changes may occur asyn-

chronously (see Note 2).

2. Materials

1. Loading of the cells with Ca

indicator and data acquisition are performed in an extracel-

lular-like, imaging medium (see Table 2 for composition), which should be prepared

fresh on the day of experimentation. Alternatively, the medium can be prepared in bulk,

sterile filtered, aliquoted, and stored at 4°C.

2. Autofluorescence and calibration media (see Table 2) are made in bulk and stored at 4°C.

3. AM esters of Ca

indicators (1 mM) should be reconstituted in dimethylsulfoxide

(DMSO), aliquoted into single-use vials, and stored at –20°C.

4. Free acids of Ca

indicators (1 mM) should be reconstituted in H

O, aliquoted into single-

use vials, and stored at –20°C.

5. Ionomycin (2 mM) should be prepared in DMSO, aliquoted into single-use vials, and

stored at –20°C.

6. Digitonin (10 mg/mL) and MnCl

(1 M) stock solutions should be prepared in H

O and

stored at room temperature.

3. Methods

3.1. Dye Loading

1. Cells should be plated onto glass (no. 1 thickness) cover slips and cultured until 70–80%

confluent in the appropriate tissue culture medium.

2. Remove tissue culture medium and rinse cover slips twice with 2–3 mL of prewarmed

imaging medium.

Principles of Ca

Imaging 445

3. Incubate cover slip with fresh imaging medium supplemented with the appropriate con-

centration of Ca

indicator and incubate with gentle agitation (see Note 3).

4. Remove medium and rinse cover slips twice with 2–3 mL of imaging medium.

5. Transfer cover slip to incubation chamber.

6. Leave for sufficient time (e.g., 10 min) to effect complete de-esterification of the dye.

3.2. Data Acquisition

1. Experiments can be performed either in a static chamber or by continual perfusion of the

cells. The former approach is recommended, because much smaller quantities of (usually

precious) materials are required. Indeed, experiments can be performed in volumes as small

as 0.3 mL.

2. Capture images for at least 60 s prior to stimulation of the cells in order to obtain an

accurate measure of the basal [Ca

] and to characterize any possible spontaneous changes

in [Ca

] (see Note 4).

3. For static chambers additions can be made by complete removal (by pipet) of the medium

and addition of fresh medium supplemented with the test agent. Alternatively, a small

aliquot of the medium can be removed, mixed with the appropriate volume of a concen-

trated stock solution of the test agent, and the entire volume added back. This method is

less prone to addition artefacts than complete exchange of the medium.

3.3. Calibration of Fluorescence Data

3.3.1. In Situ Calibration

1. This method can be used to calibrate fluorescence data from both intensiometric and

ratiometric indicators. For ratiometric indicators, autofluorescence (see below) must be

subtracted prior to calculation of the ratio.

2. At the end of the experimental run, rinse cells into imaging medium (without added Ca

)

supplemented with 2 mM BAPTA and add 2 µM ionomycin to equilibrate Ca

across the

cell membrane.

3. Reinitiate data acquisition and monitor until fluorescence intensity reaches a stable pla-

teau to obtain F

min

(for intensiometric indicators) or R

min

(for ratiometric indicators).

Table 2

Solutions for Single Cell Ca

Imaging

Imaging medium Autofluorescence medium Calibration medium

NaCl 121 mM 10 mM 10 mM

KCl 4.7 mM 120 mM 120 mM

MgCl

1.2 mM 2 mM 2 mM

CaCl

2 mM 150–300 µM —

BAPTA — 500 µM 500 µM

1.2 mM ——

NaHCO

5 mM ——

Glucose 10 mM ——

BSA 0.25% — —

HEPES 20 mM 20 mM 20 mM

pH 7.4 @ 37°C pH 7.2 @ 37°C pH 7.2 @ 37°C

446 Patel and Iozzo

4. Add 10 mM CaCl

to saturate the dye with Ca

5. Reinitiate data acquisition and monitor until fluorescence intensity reaches a stable pla-

teau to obtain F

max

(for intensiometric indicators) or R

max

(for ratiometric indicators).

6. Use eq. (1) (for intensiometric) or eq. (2) (for ratiometric indicators) to calculate [Ca

3.3.2. In Vitro Calibration

1. This method is suitable for ratiometric indicators for a given instrument configuration.

2. Place 2 mL of calibration buffer (see Table 2) in the incubation chamber and acquire

5–10 images. This is the background fluorescence, which should be subtracted from sub-

sequent data (below) at both wavelengths before calculation of ratios.

3. Add 1 µM of the free acid form of the indicator and acquire 5–10 images. Calibration

medium is initially Ca

-free, thus the fluorescence ratio gives R

min

4. Add 500 µM CaCl

to saturate the dye and acquire 5–10 images. This fluorescence ratio

is R

max

5. Use eq. (2) to convert the acquired experimental ratio values (corrected for

autofluorescence) to [Ca

] at each time point.

3.4. Determination of Autofluorescence

3.4.1. Mn

Quench

1. At the end of the experimental run, remove medium and rinse twice with 2 mL of imag-

ing buffer.

2. Reinitiate data acquisition and add MnCl

(2–4 mM) and ionomycin (2 µM).

3. Monitor until fluorescence intensity falls to a stable plateau.

4. Subtract final image from all acquired images.

3.4.2. Digitonin Permeabilization

1. At the end of the experimental run, remove medium and rinse cells twice with 2 mL of

autofluorescence buffer.

2. Reinitiate data acquisition and add digitonin (20–50 µg/mL).

3. Monitor until fluorescence intensity falls to a stable plateau.

4. Subtract final image from all acquired images.

4. Notes

1. Photobleaching and dye extrusion can be distinguished by briefly interrupting data acqui-

sition and comparing the fluorescence intensity before and after resuming acquisition. If

the fluorescent intensities are comparable, then the loss of signal is due to photobleaching,

in which case illumination intensity should be reduced by decreasing exposure time and/

or increasing attenuation of the excitation source. Dye extrusion, however, should not be

affected by stopping acquisition, thus the fluorescence signal should continue to fall.

Loss of dye can be slowed by reducing the working temperature and/or including organic

anion-transport inhibitors such as bromosulphophthalein (100 µM) in the imaging medium

to inhibit active extrusion pathways (also see Note 2).

2. Compartmentalized dye can be recognized by a punctate cellular dye distribution and/

or a dim nucleus and can be reduced by reducing the loading temperature and/or

loading time. Alternatively, the free acid (de-esterified) form of the dye can be intro-

duced directly into the cytosol by micropipet or dialysis through a patch pipet. This

method, although more disruptive, has the added advantage of allowing delivery of

dyes conjugated to inert dextrans, thereby preventing dye extrusion (see ref. 17 for

further discussion).

Principles of Ca

Imaging 447

3. The conditions for dye loading are highly dependent on cell type and should be determined

empirically. Cells are typically loaded for up to 1 hour with 1–10 µM of the AM ester. Dye

loading can be increased by premixing the dye with dispersants such as Pluronic F-127 (0.02%)

prior to dilution into imaging medium (to increase dye solubilization) and/or by decreasing

cell density. Care should also be taken not to overload cells with dye, since at high concentra-

tions, the indicator may buffer Ca

increases, resulting in sluggish or blunted responses.

4. As a rule, minimize the time that cells are exposed to the excitation light to prevent

photobleaching (see Table 1). This is achieved by adjusting shutter exposure time and

attenuation (neutral-density filter). Also, the delay between capturing successive images

(acquisition delay) will determine total exposure time. A common problem encountered

during acquisition is uneven fluorescence throughout the imaging field. This is likely to be

due to misalignment of the excitation source. Also, excitation bulbs have a finite life span.

Bulbs should be replaced if high frequency noise becomes a problem during experimentation.

Acknowledgements

We would like to thank Dr. David McQuillan for providing decorin, and G. C.

Churchill and A. Galione for helpful discussions. This work was supported by a

Wellcome Prize Travelling Research Fellowship.

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