Platelet purification and coating of plates for adhesion assays 

Blood from healthy donors was collected in 1/6th volume of acid citrate dextrose solution (ACD). Mix gently.

Blood was spun down for 20 min at 200 x g, deaccelerating step without brake.

Platelet rich plasma was collected (top yellow layer) and mixed (inverting the tube) 1:1 with HEP buffer.

Platelets were collected by centrifugation at 800 x g for 20 min without brake. At this step supernatant was removed without disturbing the pellet.

Platelets were washed twice with 1 ml platelet wash buffer as indicated: rinse the platelet pellet with

platelet wash buffer (without resuspension in order to avoid unnecessary platelet activation) by gently adding wash buffer and removing it slowly with a pipette. Repeat once. After the last wash, resuspend the pellet in 3 ml wash buffer.

Platelets were fixed with 1% paraformaldehyde for 10 minutes, incubate platelets at room temperature for 10 minutes.

Pellet platelets by centrifuging at 700 x g for 10 minutes (no brake) and wash them with 10 ml PBS pH

7.4 Repeat once.

After the second wash, resuspend platelets in PBS pH 7.4 (If we start with 180 mls of blood we typically use 10 mls here).  Count platelets and then adjust the volume of PBS to have 1x10⁷ cells/mL.

Coat flat bottom 96-well plate wells with 100 µL of platelets (1x10⁷). We coat triplicate wells for each sample and for each strain coat three control wells with 100 µL of 1% BSA.

Incubate the plate for 1h at 37°C.

Aspirate liquid and wash wells by adding 120 µL of PBS to each well. Remove liquid again and finally add 120 µL of PBS to each well.

The plates are covered in parafilm and keep in the cold room for further use. They keep at least one month