In this protocol, we propose a step-by-step tutorial for measuring platelet associated tissue factor by whole blood flow cytometry, assessing the protein in the intracellular compartment, arresting conditions and on the cell surface, both in resting condition and upon activation With a DP, one of the most common platelet agonist tissue factor is expressed by a subset of platelets. If tissue factor positive platelets are measured in PRP platelet-rich plasma, or in washed platelets, this subset can be lost during sample preparation. This risk does not occur when this evaluation is performed in wall.
Blood plate associated tissue factor has been controversial since the first report published at the beginning of the century due to pre-analytic and methodological issues. This protocol sheds light on these issues and also being used to conduct an international project supported by the International Society of Thrombosis and hemostasis ISDH to standardize platelet associated tissue factor measurement or blood flow cytometry is performed on a few microliters of blood, allowing platelet associated tissue factor analysis suitable in the clinical setting. Moreover, the possibility of fixing the sample and labeling afterwards, make this analysis feasible for many hospital and labs.
To begin, Prepare one test tube for each antibody volume. To test add one drop of negative beads and one drop of positive beads to each test tube. Add 20 microliters of each antibody dilution to the test tube and vortex.
Immediately incubate the test tubes in the dark at room temperature for 20 minutes. Then add one milliliter of PBS without calcium and magnesium pH 7.4 to each tube and vortex, gently invert the sodium citrate vacutainer five times to mix the blood and anticoagulant thoroughly. Transfer 50 microliters of blood into a 1.5 milliliter tube.
Then add 950 microliters of 1%paraform aldehyde. Gently mix and incubate the sample for 90 minutes at room temperature. Now centrifuge the sample at 1, 500 G for five minutes with a break at room temperature.
After removing the supernatant Reese, suspend the pellet in one milliliter of PBS without calcium and magnesium. At pH 7.4, dispense 100 microliters of fixed blood into each tube and centrifuge them at 1, 500 G for five minutes. With break at room temperature, then remove the supernatant resus.
Suspend the pellet in 100 microliters of 0.1%Triton PBS to perme the samples and incubate for 10 minutes at room temperature. To begin, prepare one tube for the fluorescence minus one control and one tube for tissue factor staining. Add antibodies to 100 microliters of fixed and perme whole blood in each tube.
Add 300 microliters of PBS without calcium and magnesium at pH 7.4 to each tube and mix. Then store the stained samples in the dark until flow cytometry analysis dilute alpha CD 1 42 HTF one monoclonal antibody, and Alexa Fluer 6 33 goat anti mouse immunoglobulin G in PBS without calcium and magnesium. Next, prepare three sample tubes and dispense PBS without calcium and magnesium.
At pH 7.4, dispense 7.5 microliters of diluted alpha CD 1 42 htf one monoclonal antibody, and add five microliters of adenosine diphosphate. Gently invert the citrate vacutainer five times to mix the blood and anticoagulant thoroughly. Dispense five microliters of whole blood into each tube.
Gently mix the samples and incubate for 20 minutes at room temperature. For sample fixation, add 300 microliters of 1%PFA to each tube and centrifuge at 1, 500 G for five minutes with break at room temperature. Once the supernatant is removed, resus suspend the pellet in 90 microliters of PBS without calcium and magnesium.
At pH 7.4 by gently pipetting dispense five microliters of diluted Alexa Fluer 6 33 immunoglobulin G and five microliters of alpha CD 41 PE into each tube. Incubate the samples for 15 minutes in the dark at room temperature. Then add 300 microliters of PBS without calcium and magnesium at pH 7.4 to each tube and mix.
To Begin, create a forward scatter area and side scatter area dot plot on a logarithmic scale displaying all events, create a CD 61 A and side scatter area dot plot on a logarithmic scale displaying all events. To visualize the platelet population, set the threshold to approximately 2000 on the side scatter height and 1000 on the per CP B six 90 H.Adjust the PE Y 5 8 5 and per CP B six 90 gain according to daily quality control and set the flow rate to low. Now draw a region between 10 rays to the power of three and 10 rays to the power of five called CD 61 positive.
To identify the platelet population, then duplicate the FMO and rename it TFIC. Acquire the samples and record a data file of 10, 000 events within the CD 61 positive gate. Create a forward scatter area and side scatter area dot plot on a logarithmic scale displaying all events.
Create a CD 41 A and side scatter area dot plot on a logarithmic scale displaying all events. Then adjust the Alexa floor 6 33 gain and the PE wifi 5 8 5 gain according to daily quality control. Set the threshold approximately to 1000 on side scatter height and to 2, 500 on PEY 5 85 H.To visualize the platelet population, set the flow rate to low.
Now draw a region between 10 rays to the power of four and 10 raised to the power of five called CD 41 positive to identify the platelet population. Then create a tube for each sample and name TF Unstimulated and TF Stimulated. Acquire the samples and record a data file of 10, 000 events within the CD 41 positive gait centrifuge.
The 0.129 molar sodium citrate vacutainer at 100 G for 10 minutes without break at room temperature. Then remove the vacutainer from the centrifuge. Collect the obtained platelet rich plasma, transfer it to a 10 milliliter polypropylene tube and record the volume collected count platelets twice in the platelet rich plasma using a blood cell counter.
The platelet population identified by alpha CD 61 antibody staining showed that approximately 26.8%of circulating platelets contained intracellular tissue factor flow cytometry analysis indicated that tissue factor is expressed by approximately 2.8%of resting platelets and increases to 24.5%upon activation with adenosine diphosphate. Larger sized platelets were shown to express higher tissue factor levels with the green dots representing the larger platelet subset. Flow cytometry analysis demonstrated that platelet-rich plasma prepared using 100 g centrifugation preserved a similar population to whole blood retaining larger sized platelets with higher tissue factor positivity.
Higher centrifugation force led to a loss of larger tissue factor positive platelets as indicated by the reduced forward scatter in the PRP population.
