LA IMPORTANCIA DE LOS AMBIENTES FÍSICOS INSTITUCIONALES

The flow cytometry uses the principle of light scattering in combination with light excitation/emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells in the size range of 0.5 µm to 40 µm diameter. The cells are hydro- dynamically focused in a sheath of buffer before intercepting an optically focused light source thereby producing data for each single cell. Lasers are most often used as a light source in flow cytometry. As cells or particles of interest intercept the light source they scatter light and fluorochromes are excited to a higher energy state. Fluorochromes release energy in the form of photons of light with specific spectral properties, which is unique to each fluorochrome. Scattered and emitted light from cells and particles are collected and redirected by a beam splitter to appropriate detectors, which convert the light into electrical pulses. The beam splitter splits the collected light into different wavelengths, which are routed to band pass filters of specified wavelengths that allow them to designated optical detectors. For example, a 525 nm band pass filter placed in the light path prior to the detector will only allow “green” light received by the detector. The most common type of detector used in flow cytometry is the photomultiplier tube (PMT).

Materials and methods 38

3.4.1.

Shape change measurement

In flow cytometry, the cells are characterized individually by their physical and/or chemical properties. The physical profile of cells can be observed by combining the forward light scatter (FSC) and the orthogonal or side light scatter (SSC) that are produced by cells intercepting the light beam. In forward light scatter, the intensity of laser beams uninterrupted by the cells and beams that passes around the cells, are measured. This measurement is an indication of the cell’s unique refractive index, which depends on its size, organelles, and water and molecular contents. The refractive index (FSC) of a cell can change through cell cycle progression, activation and fixation. The cellular side scatter light is the light that is reflected orthogonal to the laser beam and it is an indication of internal complexity or cell surface granularity (Figure 3.1).

Figure 3.1 Light scattering properties of a cell. The detector in the path of light source detects forward scatter light while the detector placed at 90° to the interception point detects side scatter light.

The side and forward light scatter profiles of unstimulated platelets are different from those of platelets undergoing shape change (Ruf and Patscheke 1995). During shape change, the mean FSC of platelets increases due to a decrease in average cell size, while the mean SSC decreases due to a reduction of surface granularity caused by the centralization of granules after platelet activation. Thus, the ratio of FSC/SSC increases proportionally to the extent of platelet shape change. Aliquots (100 µl) of the stimulated and unstimulated platelet suspensions were transferred to an equal volume of 0.15 M phosphate buffer (pH 7.4) containing 0.2% (w/v) glutaraldehyde for fixation. After 10 minutes, the samples were centrifuged in a microfuge (800x g for 5 minutes) and washed twice with 0.5 ml of Dulbecco’s phosphate buffered saline (PBS). The platelet pellets were resuspended in the residual volume and were incubatedfor 15 minutes in the dark at room temperature with followingantibodies: anti-CD41a–FITC antibody bindingto the glycoprotein IIb to label platelets and isotype-matched IgG1-FITC (6 µL diluted 1:100) to determine unspecificbinding. Subsequently, platelet samples were then diluted with PBS to get a concentration of platelets less than 106 cells/µl. Platelets labeled with anti-CD41a–FITCantibody and showing negative staining for IgG1-FITC isotype were gated and the mean FSC/SSC ratio of 10,000 platelet events was calculated using the CELLQuest software. The shape change induced by agonists was calculated as an increase in FSC/SSC ratio compared to that of the unstimulated platelets.

Materials and methods 39

3.4.2.

F-actin measurement using flow cytometry

Phalloidin, a toxin from the toadstool "Death Cap" (Amanita phalloides mushroom) that binds with F-actin in a stoichiometric ratio of 1:1 (Lengsfeld et al. 1974) has provided a very convenient tool to study the distribution of F-actin in permeabilized cells, since fluorescent analogs of phalloidin can be synthesized that retain its actin binding property (Wulf et al. 1979). Phalloidin binds to actin at the junction between subunits and because this is not a site at which many actin-binding proteins bind, most of the F-actin in cells is available for phalloidin labeling. In the same fixed platelet samples prepared for shape change measurement by flow cytometry, the F-actin content in resting or activated platelets was determined. The fixed platelets were permeabilized with 0.1% Triton X-100 containing 20 U/ml Alexa Fluor-546 phalloidin at 25°C and in dark for a minimum of 15 minutes. The labeled platelets were washed two times with PBS and incubated with anti-CD41a-FITC antibody as described above (section 3.4.1). The samples were resuspended in PBS (1.2 ml), and the platelets labeled with anti-CD41a-FITC were gated and analyzed for Alexa Fluor-546 phalloidin fluorescence to estimate F-actin content in these platelets. The mean fluorescence intensity of Alexa Fluor-546 from 10,000 platelet events was quantified using the CELLQuest software. The correlation of changes in F-actin content after stimulation was calculated by considering F-actin in unstimulated platelets as 40% of the total actin present in platelets.