Flow cytometric immunophenotyping

2.2.9.1 Analysis of CEACAM2 and other platelet glycoprotein expression on mouse platelets

Surface and total expression of CEACAM2 in wild-type and Cc2–/– platelets was performed using washed platelets as previously described [99]. For surface expression, 50 µL of washed platelets (100x109/L) were pre-incubated for 1 hour at room temperature with a primary antibody such as anti-rabbit CEACAM1 antibody, 2457 (1/500), anti-rabbit CEACAM2 antibody, 2052 (1/100), anti-mouse PECAM-1 (10 µg/mL), pre-immune rabbit serum (1/100), anti-mouse GPVI (10 µg/mL) and anti-mouse CD9 (10 µg/mL). The platelet-antibody complex was washed with 0.2% (w/v) BSA-RCD pH 6.5, followed by FITC-conjugated anti-rat (1/200) or PE-conjugated anti-rat (1/100) or Streptavidin-PE (1/200) for 45 minutes at room temperature. For direct antibody conjugation, platelets were pre-incubated with anti-mouse integrin β3, CD61 (10 µg/mL), anti-mouse integrin α2β1,

CD49b (15 µg/mL), anti-mouse GPIbα/IX/V, CD42b (10 µg/mL), and anti-mouse CD44 (10 µg/mL) for 45 minutes at room temperature.

To determine the total protein expression, platelets were pre-incubated with 0.1% (w/v) saponin in RCD buffer pH 6.5 and appropriate antibody for 1 hour at room temperature. The platelets were diluted by addition of 300 µL RCD buffer pH 7.4. 10,000 individual platelets were analysed on a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA) and Weasel software Version 3.0.2 (Walter and Eliza Hall Institute of Medical Research, Victoria, Australia). The template was formatted for a platelet-specific FITC/PE protocol with forward scatter (FSC), side scatter (SSC), and fluorescent parameters in log scale. The main platelet population was selected prior to the acquisition of data.

Changes to the surface expression of CEACAM1 and CEACAM2 was also measured on wild-type and Cc2–/– platelets upon addition of several agonists such as 0.125-1.0 U/mL

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thrombin, 100-300 µM PAR-4 agonist peptide and 1.0-4.0 µg/mL CRP. After addition of agonists, platelets were pre-incubated with either anti-rabbit CEACAM1, 2457 (1/500) or anti-rabbit CEACAM2 antibodies, 2052 (1/100) followed by PE conjugated anti-rat (1/200). The platelets were diluted by addition of 300 µL RCD buffer pH 7.4. 10,000 individual platelets were analysed on a FACS Canto II flow cytometer and Weasel software Version 3.0.2.

2.2.9.2 Analysis of alpha granule release (FITC-P-selectin)

Washed mouse platelets (100x109/L; 50 µL) were activated with several agonists including 0.1-0.2 U/mL thrombin, 100-300 µM PAR-4 agonist peptide and 0.5-2.0 µg/mL CRP or CLEC-2 selective agonist, Rhod at 0.6-1.2 µg/mL for 15 minutes at 37°C. The activated platelets were fixed with 1% (w/v) paraformaldehyde for 10 minutes at room temperature, and then terminated with RCD pH 6.5. Fixed platelets were then pre-incubated with 10 µg/mL P-selectin antibody (FITC-CD62P) for 30 minutes at room temperature, then washed with 0.2% (w/v) BSA-RCD pH 6.5, then spun at 640 g for 10 minutes without brake. The platelets were diluted by addition of 300 µL RCD buffer pH 6.5. Alpha granule exocytosis was identified using platelet population characteristic forward and side laser scatter and examined for expression of FITC-CD62P. 10,000 individual platelets were analysed on a FACS Canto II flow cytometer and Weasel software Version 3.0.2 [219].

2.2.9.3 Analysis of dense granule release using quinacrine uptake

Washed mouse platelets (100x109/L; 50 µL) were pre-stained with 100 µM of 500 µM quinacrine dye for 30 minutes at 37°C. Stained platelets were then terminated with 0.2% (w/v) BSA-RCD pH 6.5, and then spun at 640 g for 10 minutes without brake. The platelets were activated with several agonists including 0.125-1.0 U/mL thrombin, 100-300 µM PAR-4 agonist peptide and 0.25-4.0 µg/mL CRP or 0.4-1.2 µg/mL Rhod of CLEC-2 selective agonist, for 10 minutes at 37°C. The platelets were diluted by addition of 300 µL

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RCD buffer pH 7.4. Dense granule exocytosis was recorded as the percentage decrease in quinacrine fluorescence intensity compared to resting platelets. 10,000 individual platelets were analysed on a FACS Canto II flow cytometer and Weasel software Version 3.0.2.

2.2.9.4 Analysis of soluble FITC-fibrinogen and JON/A-PE binding

FITC-conjugated fibrinogen was prepared by conjugating 20 mg human fibrinogen to FITC (Sigma Chemical, St Louis, MO) in 0.15 M carbonate buffer pH 9.0 for 1 hour at room temperature. The FITC-labelled fibrinogen was purified using a PD10 column (Amersham Pharmacia, Piscataway, NJ), and then the column was eluted with 15 mL (15 x 1 mL) of PBS and 1 ml fractions were collected [8, 220].

Measurement of FITC-labelled fibrinogen and PE-conjugated JON/A mAb binding to platelets via GPIIb/IIIa (integrin αIIbβ3) was performed. Washed mouse platelets (50 µL

containing 100x109/L in RCD pH 6.5) were pre-incubated with FITC-labelled fibrinogen at 1.2 µg/mL or PE-conjugated JON/A mAb (1:50 dilution) in the presence or absence of agonists. Platelets were activated with agonists including 0.125-1.0 U/mL thrombin, 100- 300 µM PAR-4 agonist peptide, 10 µM ADP, mixture of 10 µM ADP plus 20 µM Epi, 20 µM PMA and 0.25-4.0 µg/mL CRP for 1 hour at 37°C in a dark room. Washing steps were performed using RCD pH 6.5 containing 0.2% (w/v) BSA after each step. The platelets were fixed with 1% (w/v) paraformaldehyde for 10 minutes at room temperature. They were then diluted by addition of 300 µL RCD buffer pH 6.5 and 10,000 individual platelets were analysed as described in section 2.2.9.1.

2.2.9.5 Phosphatidylserine exposure

PRP and washed platelets were generated as described in section 2.2.2. PRP was pre- incubated with 1 mM acetylsalicylic acid for 45 minutes at 37°C. Washed platelets were isolated and the pellet was gently resuspended in 500 µL Tyrode’s buffer (10 mM HEPES, pH 6.5, containing 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM

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glucose) containing 0.1 U/mL apyrase. 100x109/L washed platelets were pre-incubated with 2.5 mM CaCl2 and 2 µL of FITC-labelled Annexin V, followed by agonist stimulation,

0.125-0.5 U/mL thrombin, 100-300 µM PAR-4 agonist peptide and 1-4.0 µg/mL CRP for 45 minutes at 37°C. The labelled platelets were then terminated with 0.2% (w/v) BSA- Tyrode’s buffer pH 6.5, and then spun at 640 g for 10 minutes without brake. The platelets were diluted by addition of 300 µL Tyrode’s buffer and 10,000 individual platelets were analysed on a FACS Canto II flow cytometer and Weasel software Version 3.0.2.