Platelet Adhesion and cell rolling assays protocols

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Coating the plate with collagen (for use with low shear plates 0-20 dyne)

Preparation of 40ug/mL collagen in 20 uM 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 20uM acetic acid in a tube. Add 40uL of collagen (1mg/mL) 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 at least 1 hour at room temperature.

Washing and blocking the channels with BSA

  1. Remove excess fluid from the outlet well but do not allow the well to dry (empty well will introduce bubbles into the channel)
  2. Add 500uL PBS containing 0.5% BSA to the outlet wells. Remove any bubbles that may form by pipetting or gently move the tip around to dislodge the bubbles
  3. Apply 5 dyne/cm for 15 minutes to the outlet wells
  4. Remove any excess PBS containing 0.5%BSA 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. Plates may be stored at 4°C overnight.

Preparation of platelet rich plasma (PRP)

Blood should be collected fresh from healthy individual and process same day for experiment.

  1. Centrifuge the whole blood at 180 X g for 15 minutes. Remove the supernatant PRP (~2mL, top portion) into new tubes.
  2. Centrifuge the PRP again at 20 x g for 5 minutes to remove any remaining leukocytes. Remove and use only the top "clean" PRP portion. Discard the pellet containing leukocytes.

Labeling PR with Calcein AM:

  1. Lyophilized Calcein AM (Sigma 56496-20x50UG) comes in 50 µg aliquots. (Tip: Thaw a fresh Calcein AM tube from the -20°C when the blood goes in centrifuge for first spin.)
  2. Briefly spin the Calcein AM tube to bring down the content to the bottom of the tube.
  3. Dissolved the entire content (50ug; MW = 994.87 g/mol) in 50 uL DMSO to make a 200x stock concentration of 1mM.
  4. Dilute PRP with HBSS or platelet poor plasma (PPP) to make normalized 15,000platelet/ul for every sample.
  5. Use 5uL 1mM Calcein AM for every 1mL of normalized PRP.
  6. Incubate for at least 30 minutes. The labeled PR can now be used for experiment.

Running the platelet adhesion experiment

  1. 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. 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 labeled 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, bright field, 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 under FITC channel and start image acquisition

Coating channels with vWF for platelet rolling experiment

  1. Add 100 pL 50 to 100ug/mL von Willerbrand factor (vWF) 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 [Start]. 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 1 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/cm- 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 adhesion 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/cm2 for 10 minutes
  5. Remove the whole blood and rinse the inlet wells with PBS
  6. Add 500L of new HBSS and 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/cm2 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) under bright field.