Flow cytometric analysis of mouse platelet surface glycoproteins

           - Preparation of diluted or washed whole blood  

           - Preparation of washed mouse platelets  

           - Single-color analysis of platelet surface glycoprotein expression 

           - Two-color analysis of platelet activation

  Immunohistochemistry (with acetone-fixed frozen sections)

  Immunoprecipitation

  Immunoblot (Western Blot Analysis)

Buffers and reagents

Tris buffered saline/Heparin (TBS/Hep): 20 mM Tris/HCl; 137 mM NaCl; pH 7.3; containing 20 U/ml Heparin.

Phosphate buffered saline (PBS): 137 mM NaCl; 2.7 mM KCl; 1.5 mM KH2PO4; 8 mM Na2HPO4; pH 7.14.

Tyrode’s Buffer (modified): 134 mM NaCl; 0.34 mM Na2HPO4; 2.9 mM KCl; 12 mM NaHCO3; 20 mM Hepes; pH 7.0; 5 mM glucose; 0.35% (w/v) bovine serum albumin

Apyrase: 10 U/ml stock in H2Obidest (stored at -20°C)

Prostacyclin (Prostaglandin 2, PGI2): 1 mM stock in H2Obidest (stored at -20°C)

CaCl2: 1 M stock in H2Obidest

Preparation of diluted or washed whole blood

1. Take 50 μl blood with a heparinized glass capillary (e.g. ringcap) from the retro-orbital plexus into 1.5 ml tubes containing 200 μl of TBS/Heparin (20 U/ml).

2. Dilute in 1 ml of Tyrode′s buffer and use for flow cytometric analysis. 

3. To prepare washed blood, centrifuge the diluted blood at 900 x g for 5 min, remove the supernatant and resuspend the pellet in 1.25 ml Tyrode′s buffer.

4. Add 1 mM CaCl2 directly before the start of the experiment.
   

Note: For thrombin-induced platelet activation, plasma has to be removed to prevent anti-thrombin activity

Preparation of washed mouse platelets

The preparation of washed platelets is a more time consuming method, that requires a larger amount of blood, but yields a platelet preparation that can be used for several hours.

1. Collect 0.5 - 1 ml blood with 1.5 cm glass capillaries from the retro-orbital plexus into a 1.5 ml tube containing 200 μl of TBS/Heparin (20 U/ml).

2. Centrifuge the sample for 5 min at 500 x g and transfer the platelet rich plasma (PRP) into a new tube. For best recovery of platelets, take the complete white phase including some red blood cells.

3. Centrifuge the platelet suspension for 8 min at 300 x g, and transfer the PRP without any red blood cells into a new tube.

4. Add 0.5 μM prostacyclin (PGI2) and centrifuge at 1300 x g for 5 min.

5. Resuspend the platelet pellet in 1 ml Tyrode’s buffer, add 0.02 U/ml apyrase and 0.5 μM prostacyclin, incubate for 5 min at 37°C, and centrifuge for 5 min at 1300 x g.

6. Repeat step 5 and resuspend the platelet pellet in 0.5 ml Tyrode’s buffer, add 0.02 U/ml of apyrase and incubate for 30 min at 37°C.

Single color analysis of platelet surface glycoproteins

1. Combine 5 μl of specific or negative control antibodies and 25 μl diluted whole or washed blood in the assay tube and vortex mix for 1-2 seconds.

2. Incubate for 15 min at room temperature.

3. Stop reaction by addition of 400 μl PBS and analyze within 30 min.

Two color analysis of platelet activation

1. Add 5 μl specific or negative control antibodies to the assay tube together with 5 μl of a pan-platelet marker.

2. At this point, any additional reagents (e.g. inhibitors) are added in small volumes (< 5 μl).

3. Add 26 μl diluted whole or washed blood or washed platelets (1 million) and vortex mix for 1-2 seconds.

4. Incubate for 15 min at room temperature. Stop reaction by addition of 400 μl PBS and analyze within 30 min.

Immunohistochemistry (with acetone-fixed frozen sections)

Please note: It is not recommended to perform immune staining on (para-)formaldehyde-fixed samples, since most rat antibodies yield only poor results under these conditions on mouse tissues.
 

1. Mount sections from frozen tissue on slides and fix in ice cold 100% acetone for 20 min at 4°C. Dry samples over night at RT.

2. To minimize reagent volumes, a hydrophobic ring may be drawn with a water repellent pen around the tissue section on the slide. During all following procedures, this encircled area should not dry up. Therefore, it is recommended to perform incubation steps in a humid box.

3. Incubate slides in PBS for 20 min.

4. When using peroxidase-conjugated secondary antibodies, incubate slides with 0.03% H2O2 for 10 min at RT to inhibit endogenous peroxidase activity. Rinse slides three times in PBS for 5 min each wash.

5. Incubate slides for 1 hour at RT in blocking solution (serum from host species of secondary antibody to be used), diluted 1:10 or 1:100 in PBS.

6. Cover slides with primary antibody (2-10 μg/ml) in blocking solution and incubate for 2 h at RT.

7. Blot excess liquid from slides and rinse three times in PBS for 5 min each wash.

8. Cover slides with fluorophore- or peroxidase-conjugated secondary antibody (e.g. affinity purified donkey anti-rat IgG-FITC or -HRP, Jackson ImmunoResearch, West Grove, PA) diluted in blocking solution according to the manufacturer’s instructions. Incubate for 1 h at RT.

9. Blot excess liquid from slides and rinse three times in PBS for 5 min each wash.

10. a) Fluorescently stained samples can be directly analyzed by fluorescence microscopy.

11. b) For visualization of peroxidase-labeled structures, cover slides with freshly prepared 3-amino-9-ethylcarbazole (AEC) substrate and incubate until red staining is visible under the microscope, time can vary between 5 and 30 min. Stop reaction by rinsing with deionized water before orange background staining occurs. For counterstaining, incubate samples for 30-240 s with hematoxylin and rinse slides for 20 min with running warm water. Mount sections with aqueous embedding solution (e.g. Aquatex, Merck).

Buffers and Reagents

PBS/EDTA: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.0 mM Na2HPO4 x 2 H2O, pH 7.3; 5 mM EDTA

IP buffer: 40 mM Tris/HCl, 0.3 M NaCl, 1 mM EDTA, 2 mM PMSF, 10 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 μg/ml Pepstatin

Igepal: 10% stock in H2Obidest

2x SDS-sample buffer: 10% beta-mercaptoethanol (for reducing buffer), 10% Tris buffer (1.25 M), 20% Glycerin, 4% SDS, 0.02% Bromophenolblue 

1. Wash mouse platelets or cells (10 million per immunoprecipitation) 3 x in1 ml PBS/EDTA (5 min, 1300 x g) and resuspend in IP-buffer.

2. Lyse platelets by addition of 1% Igepal for 15 – 20 min at RT.

3. To remove cell debris, spin 5 min at 10,000 x g and transfer supernatant to a new cup.

4. Preclear by adding 20 μl of washed protein G agarose (PGA). Incubate with rotation for at least 3 h at 4°C.

5. Centrifuge the samples for 5 min at 3000 x g. Fill supernatants into new tubes, add the antibody at a concentration given in the data sheet (usually between 2 and 5 μg/ml), and after 30 min add 25 μl of washed PGA. Incubate with rotation for at least 2 h at 4°C, preferably over night.

6. To wash the precipitate, centrifuge the samples for 1 min at 3000 x g. Add 1 ml of 1 x IP buffer containing 1% NP-40 to the PGA pellet. Centrifuge for 1 min at 3000 x g.

7. Wash the PGA pellet twice in plain IP-buffer (1 min at 3000 x g).

8. Resuspend the PGA pellet in 25 μl of 1x SDS-sample buffer (reducing or non-reducing) and boil for 5 min. The samples can then be frozen (-20°C) for later use, or SDS-PAGE and subsequent immunoblot analysis can be performed.

Buffers and reagents

Lysis buffer: 40 mM Tris/HCl, 0.3 M NaCl, 1 mM EDTA, 0.05% NaN3, 1% NP-40

PBS: 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.0 mM Na2HPO4 x 2H2O, pH 7.3

PBS-5% milk: PBS containing 5% non-fat dry milk

PBS/T: PBS containing 0.1% Tween 20

PBS/T-1% milk: PBS/T containing 1% non-fat dry milk

All steps can be performed at room temperature:

1.     Prepare lysate of mouse platelets or cells in lysis buffer and incubate with (2x) SDS sample buffer for 5 min at 95°C.

2.     Separate proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions, as indicated in the respective data sheet, and transfer the proteins to PVDF membrane.

3.     Block the membrane with PBS-5% milk for 1 h with agitation.

4.     Incubate the membrane with 2-5 μg/ml of primary antibody in PBS/T-1% milk for 1 h with agitation.

5.     Rinse the membrane twice with PBS/T followed by two 30 min washing periods in PBS/T. Washing procedure may be reduced to three periods of 10 min each, but that should be determined individually.

6.     Incubate with secondary antibody (HRP-conjugated anti-rat Ig, e.g. from DAKO, Glostrup, Denmark, #P0162) in PBS/T-1% milk

for 45–60 min with agitation.

7.     Rinse the membrane twice with PBS/T followed by two 30 min washing periods in PBS/T.

8.     Visualize bound antibody with ECL (enhanced chemiluminiscence).