Platelet isolation and quantification

Materials and Buffers:

Heparinized capilars

Tyrodes buffer: 134 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, 10 mM N-2-hydroxyethylpiperazine-N-2- ethanesulfonic acid, 5 mM glucose, 0.35% BSA. pH adjusted with HCl

20U/ml Heparin (diluted in autoclaved water)

Procedure:

1.      Collect blood from mice using Eppendorf tubes containing 300-400 µl Heparin (20U/ml)

2.      Centrifuge (soft mode) at 700 rpm (70 xg) for 10 min at RT

3.      Collect the supernatant carefully in a new Eppendorf.

4.      Add more heparin to pellet and centrifuge again as in 2.

5.      Collect supernatant carefully and, if wanted, mix it with the previous one or place it in a new tube.

6.      Centrifuge in soft mode supernatant 2500 rpm during 7 min at RT

7.      Remove supernatant and resuspend pellet (white) with 1 ml Tyrodes buffer pH6.5 to wash it

8.      Centrifuge in soft mode supernatant 2500 rpm during 7 min at RT

9.      Resuspend in 150-250 µl Tyrodes buffer pH7.5

Keep platelets at 30°C for max 30 min while quantifying them.

10.    Measure number platelets/ml using a ProCyte Hematology Analyzer (IDEXX Laboratories)

To isolate platelets, heparinized blood extracted from Itgb3+/+ or Itgb3-/- mice was centrifuged at 70 x g for 10 min at RT. The platelet enriched upper phase was then centrifuged at 800 x g for 10 min and the platelet pellet was finally washed twice with Tyrodes buffer pH 6.5. Washed platelets were resuspended in Tyrodes buffer pH 7.4 and counted using a ProCyte Hematology Analyzer (IDEXX Laboratories). For experiments, platelet numbers were adjusted to equivalent concentrations with Tyrodes buffer pH 7.4 complemented with 1 mM CaCl2, 1 mM MgCl2.