Platelet Adhesion and Rolling - Mouse Blood Protocol

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Platelet adhesion protocol (mouse blood)

Coating the plate with collagen

Preparation of 40ug/mL collagen in 20uM acetic acid and coating the channels

  1. Make sure the plate and coating material are same temperature to reduce bubble formation
  2. Allow the collagen (Chronolog P/N 385 1mg/mL) to reach ambient temperature
  3. Centrifuge at 3000xg for 2 minutes. Use only the top portion
  4. Place 960uL of 20 uM acetic acid in a tube. Add 40uL of collagen (1ug/uL) and pipette up and down gently to mix. Working concentration is 40ug/mL
  5. Add 100uL 40ug/mL collagen to outlet wells. Dislodge any bubbles. Clamp on the interface
  6. Under the Bioflux manual mode, highlight the appropriate column of the wells (select the outlet columns) and press [Pause]. Apply 10 dyne/cm and press [Start]. Press [Stop] after 30 seconds.
  7. Remove the interface and check to make sure there is a small droplet in each inlet wells. This indicates that the collagen solution had gone through. Check the channel viewing areas for air bubbles. Dislodge as necessary
  8. Incubate for 1 hour at room temperature

Washing and blocking the channels with BSA

  1. Add 500uL 0.5% BSA/PBS to the inlet wells. Remove any bubbles that may form by pipetting gently to dislodge the bubbles
  2. Apply 5 dyne/cm2 for 15 minutes to the inlet wells
  3. Remove any excess BSA/PBS from the inlet and outlet wells. Coated plate is now ready to use. Add 200uL PBS to the inlet and outlet wells if plates are not to be used immediately

Preparation of mouse platelet rich plasma (PRP)

  1. Collect mouse blood by cardiac puncture into a syringe containing ACD (2.5% Sodium citrate, 2% D-glucose and 1.5% citric acid) at ratio of ACD to blood of 1:6
  2. Centrifuge the blood at 2300x g for 20 seconds in a fixed rotor centrifuge at room temperature
  3. Transfer the top plasma portion containing PR into new tubes
  4. Add PGI2 to final concentration of 0.5 uM. Centrifuge at 2200x g for 3 minutes
  5. Wash once with Tyrodes-HEPES containing 0.5uM PGI2. Remove and discard supernatant
  6. Re-suspend the platelet pellet in Tyrodes-HEPES containing 0.5 uM prostaglandin I2 (PGI2) and 10 U/mL Heparin to 10-15,000 platelets/uL
  7. Add CellMask Green to 1X working concentration and allow the platelets to rest for 30 minutes before performing aggregation assays

Running the platelet adhesion experiment

Program the auto-run starting at 2 dyne/cm- 20 second, and then increase to 30 dyne/cm- or to the desired shear rate for 10 minutes. Save the Bioflux run sequence


Image Acquisition

  1. If necessary remove PBS from inlets and outlets wells of the coated channels
  2. Add 200uL PBS to outlet wells (for balance). Mix by inverting gently the PRP tube. Add 300uL PRP to inlet wells. Clamp on the interface.
  3. Put plate in holder and put on microscope stage
  4. Under Bioflux manual mode, highlight the inlet wells and perfuse at 1 dyne/cm2
  5. Under 10X objective, find and focus the desired channel(s)
  6. Under Bioflux Auto Run mode, start the saved Bioflux run sequence (the perfusion profile
    programmed previously)
  7. Program the image acquisition at every 20 seconds for the duration of the aggregation profile
  8. Start acquisition

Material and reagents required

  • Bioflux plate (24 or 48-well plates)
  • 20 uM acetic acid in water
  • Collagen (Chronolog P/N 385)
  • 0.5% BSA in PBS (Ca2+ Mg?+-free)
  • PGI2, a platelet activation inhibitor
  • Tyrodes-HEPES or HBSS
  • Cell counter
  • DiOC6, a green-fluorescent membrane dye for staining mouse platelets
  • Willerbrand factor (WF) solution

Platelet Rolling Protocol (mouse blood)

Coating channels with WF for platelet rolling experiment

  1. Add 100uL 50 to 100ug/mL von Willerbrand factor (WF) solution to outlet wells. Dislodge any bubbles. Clamp on the interface
  2. Under the Bioflux manual mode, highlight the appropriate column of the wells (select the outlet columns) and press [Pause]. Apply 10 dyne/cm2 and press [Pause] again to un-pause. Press [Stop] after 30 seconds.
  3. Remove the interface and check to make sure there is a small droplet in each inlet wells. This indicates that the WF solution had gone through. Check the channel viewing areas for air bubbles. Dislodge as necessary
  4. Incubate for 1 hour at room temperature
  5. Add 500uL 0.5% BSA/PBS to the inlet wells. Remove any bubbles that may form by pipetting gently to dislodge the bubbles
  6. Apply 5 dyne/cm2 for 10 minutes to the inlet wells
  7. Remove any excess BSA/PBS from the inlet and outlet wells. Coated plate is now ready to use. Add 200uL PBS to the inlet and outlet wells if plates are not to be used immediately

Running the platelet rolling experiment

Load channel with platelets from whole blood

  1. If necessary remove PBS from inlets and outlets wells of the coated channels
  2. Add 200uL PBS to outlet wells (for balance). Mix by inverting gently the whole blood tube. Add 300uL to inlet wells. Clamp on the interface.
  3. Put plate in holder and put on microscope stage
  4. Under Bioflux manual mode, highlight the inlet wells and perfuse at 30 dyne/cm- for 10 minutes
  5. Remove the whole blood and rinse the inlet wells with PBS
  6. Add 500uL of new HBSS and perfuse at 10 dyne/cm- for 5 minutes or until all WBC is clear of the channel

Image acquisition

  1. While under flow, find and focus the desired channel(s) using the 10X objective
  2. Under Bioflux Auto Run mode, program a shear rate of 60dyne/cm- for 15 minutes. Save the
    Bioflux run sequence.
  3. Start the saved Bioflux run sequence
  4. Stream and record the rolling of platelet or acquire image every second for the duration of the desired rolling time (2-5 minutes)