Flow cytometric analysis

5.1. Staining of cell surface antigens

Cells were detached by rinsing Ultra Low Attachment Surfaces with PBS. After washing with PBS (w/o Ca2+, Mg2+) cells were resuspended in PBS (w/o Ca2+, Mg2+), 0,5% BSA at 5x10⁵/100 µl and incubated for 15 minutes on ice with saturating concentrations of the antibodies. Antibodies used for detection were directly fluorochrome-conjugated or indirectly labelled by subsequent incubation with fluorochrome-conjugated secondary reagents after two washing steps with PBS, 0,5% BSA.

5.2. Staining for intracellular immunofluorescence

Cytoplasmatic antigens were detected using a modified version of Jung´s staining protocol.

5x10⁵ monocytes were incubated in 500 ml fixation buffer (PBS (w/o Ca ²⁺, Mg²⁺), 4% (w/v) formaldehyd) for 15 min on ice. Cells were washed twice with permeabilization buffer (PBS

(w/o Ca ²⁺, Mg²⁺), 1% (v/v) fetal calf serum, 0.1% (w/v) saponin, 0.1% (w/v) sodium azide) and labelled with primary antibodies for 15 min on ice followed by two washing steps with PBS (w/o Ca ²⁺, Mg²⁺), 0.5% BSA. In case of unconjugated or biotin-conjugated primary antibodies labelled cells were washed twice with permeabilization buffer and incubated with the respective secondary reagents for 15 min on ice followed by two further washing steps with PBS (w/o Ca ²⁺, Mg²⁺), 0.5% BSA.

5.3. Analysis of stained cells

The cellular light-scatter signals and three fluorescence signals of 20 000 cells per sample were analyzed in list mode at a channel resolution of 1024 with forward scatter as the trigger parameter on a FACSCalibur flow cytometer (excitation: 488 nm (argon) / 635 nm (diode);

emission filters: 530/30 nm band pass (channel 1) / 585/47 nm band pass (channel 2) / 670 nm long pass (channel 3): 661/16 nm band pass (channel 4) (Fig.18); data acquisition was performed by Cellquest software (Becton Dickinson, Heidelberg, Germany). The photomultiplier gains were calibrated with polychromatic fluorescent reference beads (Polysciences). Compensation was adjusted with fluorochrome-coated microbeads (Becton Dickinson, Heidelberg, Germany). Gating of cultivated monocytes/monocyte-derived macrophages (MDM) was based on forward- and side-scatter dot plots as well as CD14 and CD45 antibodies. Mean fluorescence values were corrected for background by substraction of mean fluorescence of cells without antibody respectively if incubated with secondary antibody substraction of mean fluorescence in absence of the primary antibody.

For analysis of the list files the software CellQuest 2.0 (Becton Dickinson, Heidelberg, Germany) on a Macintosh G3 was used.

The mean and standard deviation of the mean fluorescence intensities of the independent experiments were calculated. Statistical significancy was tested by paired student´s t-test.

For analysis of surface membrane receptors during E-LDL and Ox-LDL loading, FACSCanto flow cytometer (BD Biosciences) the successor to FACSCalibur was used. It has two lasers, similar to the FACSCalibur (488nm and 633nm), however, the instrument was upgraded to detect 6-colors simultaneously (4-colors off the 488nm and 2-colors off the 633nm plus forward and side scatter). The FACSCanto has digital electronics and an octagon detection system. Data of these experiments were analyzed by FACSDiVa and CellquestPro softwares (both BD Biosciences).

5.4. Apoptosis-test with AnnexinV-FITC and Propidiumjodid (PI)

In the early stages of apoptosis, translocation of phosphatidylserine (PS) from the inner part of the plasma membrane to the outer layer takes place, by which PS becomes exposed at the external surface of the cell (373). AnnexinV is a Ca²⁺ dependent phospholipid-binding protein with high affinity for PS and is therefore suited to detect apoptotic cells. The simultaneous DNA stain with PI allows the discrimination of necrotic cells (Annexin V and PI positive) from apoptotic cells (Annexin V positive and PI negative).

Staining procedure:

After harvesting cells by gently rinsing costar plates with PBS, cells were washed twice with PBS and resuspended in AnnexinV Binding Buffer at a concentration of 1x10⁶ cells/ml. 100 ml of this solution (1x10⁵ cells) were transferred to a 5 ml tube and 5 ml of AnnexinV-FITC and 5 ml of PI were added. Cells were gently vortexed and incubated at room temperature for 15 min in the dark. After adding 400 ml of AnnexinV Binding Buffer, samples were analyzed on a flow cytometer using 488 nm excitation and a 515 nm band-pass filter for fluorescein detection and a filter >600nm for PI detection. Ustained cells as well as cells stained with AnnexinV-FITC alone and cells stained with PI alone were used as controls to set up compensation and quadrants.

6. Microscopy

Cells were cultured in LAB-TEK borosilicate chamber slides, washed with PBS, fixed in 4%

paraformaldehyde for 15 min, quenched with 50mM NH₄Cl/glycin for 15 min and then blocked with PBS/0.5% BSA for 15 min at RT.

For analysis of raft microdomains cells were incubated for 30 min at 4°C with saturated concentration of lipid vesicles in PBS, prepared from a mixture of

1-palmitoyl-2-oleyl-sn-Figure 18: Principle of a FACS Calibur Flow Cytometer

glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine-tetramethyl-rhodamine (DMPE-TMR) at a ratio of 100:1 by sonication for 30 min as described by Schütz G. J. et al. (329). Afterwards cells were washed extensively with PBS and analyzed by confocal microscopy.

For differential raft analysis, cells were incubated with anti-ceramide mAB 15B4 (1:20) for 3h at 4°C and then labeled with goat anti mouse-Cy5 secondary antibody (1:150) for 1 h at 4°C, followed by incubation with theta-toxin-FITC (1:2000) for 20 min at 4°C for cholesterol staining or cholera toxin for GM1 ganglioside staining.

For sphingomyelin staining cells were incubated with lysenin (1:100) for 4h at 4°C.

Afterwards anti-HIS antibody (1:150) for binding to the HIS of lysenin was added for 2h at 4°C followed by labelling with anti-rabbit texas-red antibody (1:150) for 30 min at 4°C.

Confocal images were acquired with an inverted TCS-4D confocal microscope (Leica, Heidelberg, Germany) equipped with a ArKr 75 mV mixed-ion laser (Melles Griot, Omnichrome, Carlsbad, CA, USA) and observed with an oil immersion objective 100x lens.

Images were acquired with a 488 nm laser line (excitation) and detected with a 535/40m band pass filter (emission). Acquisition was performed with the Scan Ware software package of the instrument and analysis was done using the software Metamorph (Universal Imaging, Corp., PA, USA).

Fluorescence images were taken with HCS System Discovery-1 (Universal Imaging, Downingtown, PA, USA) which is a conventional high-end flourescence inverse microscope with 6-fold ojective-revolver consisting of a multiSpec-imager for emission beam splitting and a high end CoolSNAP HQ camera. The system is controlled by a high-end MetMorph imaging system for measurements and data handling.

The C-terminal domain(domain 4) of the -toxin (perfringolysin O) was synthesized by the biotechnology workgroup of the Center of Excellence for Fluorescent Bioanalysis (KFB, Regensburg, Germany) according to Shimada et al 2002 (374). FITC N-terminus labeling was carried out by a standard procedure, yielding in a product with absorbance and emission maxima of 490 nm and 520nm, respectively.