Platelet Adhesion Assay- CalceinAM labeled Whole Blood

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Warning: Before beginning this experiment, please be aware that when operating at shears above 10 dynes, fluid in the wells will be depleted rapidly. It is best to set an audible alarm/timer for 10 minutes after you begin flow to ensure that you do not run the channel dry.

Priming the Channels

Experiment is outlined for setup in BioFlux 48-well plate (900-0013) and BioFlux 48-well high shear plate (900-0017). If setup is desired in BioFlux 24-well plate (900-0014) for longer run time, double all volumes where appropriate.
Before using the BioFlux Plates, it is important to prime the channels, which will be used in your experiment. Priming is simply a step to introduce fluid all the way through the channel and serves as a way to prevent air bubbles from getting in to the system.

To prime a channel, introduce a small volume (≤200 uL) of a "compatible" liquid (i.e. media, PBS, etc.) into the outlet wells). Connect the interface and apply a lower shear force (≤5 dyn/cm?) for up to 5 minutes to push the fluid up into the inlet wells. You should observe a small drop of liquid in the inlet wells. When priming channels of interest, it is important to inspect channels after priming to make sure that the entire path has been filled with liquid.

Table 1

Ligand Coating the Channels

BioFlux Plate channels can be coated with a variety of reagents for the purposes of promoting cell adhesion or to induce an interaction between the coating and subsequent introduction of cells. The coating reagent can be introduced into the outlet well(s) and can function as the priming step. Once the channel is coated, you may follow the recommended protocols or manufacturer's guidelines for adherence. Often there is a recommended waiting time for the coating to adhere (i.e. 1 hour) as well as a recommended temperature. If the volume of the coating reagent used is less than 50 uL and there is only a small droplet of fluid in the inlet well, it is recommended to use the adhesive film provided to prevent evaporation of the fluids.

In most cases, you may coat and prime the channels simultaneously

  1. Coat/prime with appropriate coating for your experiment and incubate for recommended time - usually 1 hour at room temperature according or according to the manufacturing's suggestions. Add coating at desired concentration to the outlet well, see table 1 for minimum and recommended volumes and perfuse between 2-5 dyn/cm until you see a small bead of liquid in the lower inlet well. If using recommended volumes continue perfusion until the inlet lower well is filled with coating solution. Incubate for prescribed time and at recommended temperature according to manufacturer's instructions.
  2. Add 500 ul to outlet well of pre-warmed medium or buffer and wash coated channels at 5 dyn/cm2 for 10 minutes (follow manufacturer's suggestion for washing).

Platelet Activation Via Collagen 1

Preparation of the plate with Chronolog collagen I (Cat# 385) coating:

  1. Dilute collagen 1 to 100 g/mL (range of 10-200 ug/mL) in a 0.02M acetic acid or 0.01M HCL solution in water. Add 200 ul of collagen solution to the outlet well and perfuse at 5 dyn/cm^ for 5 minutes. Stop flow and incubate at room temperature for 1 hour.
  2. Aspirate excess collagen solution from inlet and outlet wells and add 500 uL of PBS containing 0.5% BSA to the outlet well and wash at 5 dyn/cm- for 15 minutes.
  3. Remove wash solution from all wells and add 500 uL of PBS to all inlet wells and 1 mL of PBS to all outlet wells for BioFlux 48-well plate If (900-0013). If using BioFlux 48-well high shear plate (900-0017) reverse the volumes for the inlet and outlet respectively. The plate is ready to use and can be kept at room temperature until use or stored at 4°C if prepared under sterile conditions.

Once the plate is ready, proceed with the blood preparation.

  1. Draw blood into anticoagulant. If you are using sodium citrate, EDTA or heparin proceed to step b. if you are using CTI alone at 60 ug/mL to 250 mg/mL you will have to complete the experiment within 10 minutes, where 5 minutes is allocated for setup and 5 minutes experimental flow time. Using CTI prevents the use of calcein AM , instead label platelets with a specific surface antigen with direct fluorescence-conjugated antibody.
  2. Label whole blood with 10 uM calcein AM for 20 to 30 minutes at room temperature. Note, this labeling method will not work with the anticoagulant CTI. Add the calcein AM to the whole blood and very gently invert tube a few times to mix. Use immediately when incubation is complete.
  3. When using BioFlux 48-well plate (900-0013) perfuse blood in from outlet well, whereas if using BioFlux 48-well high shear plate (900-0017) perfuse blood from inlet well. Begin by removing PBS from respective wells and add 100 ul to 1 mL of blood to plate appropriate initiation well. Note if volume of blood exceeds 500 L remove PBS from both inlet and outlet side. Assay specific shear ranges from 10 to 200 dyn/cm?. Coordinate image capture accordingly using fluorescence  microscopy in the FITC range. It is best to begin time-lapse capture on a region of interest immediately as you begin flow and continue capturing until the desired outcome is achieved.
  4. Alternatively, you can set an end time for the experiment and capture data once flow is halted and image collagen induced thrombi under these stable conditions.

This protocol may be used as a general guideline for platelet activation assay. However, optimal shear stress to observe platelet activation needs to be determined empirically if other coating substrates are used, such as von Willebrand factor, fibrinogen, tissue factor. For additional coating substrates refer to Fluxion Biosciences "Experimental Protocols for Cell Monolayers, Table 2"