Chapter 28

Flow Cytometry Protocols for Assessment of Platelet

Function in Whole Blood
Leonardo Pasalic, Gabrielle J. Pennings, David Connor,
Heather Campbell, Leonard Kritharides, and Vivien M. Chen

Abstract
Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers
of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based pro-
tocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose
inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional
responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium
flux, and phosphatidylserine exposure.

Key words Platelet function, Flow cytometry, Whole blood, Glycoprotein receptor deficiency, Dense
granule release, Procoagulant platelets, Calcium flux, Platelet-leukocyte aggregates

1  Introduction

Platelets play a pivotal role in the normal hemostatic process and

pathological thrombosis. Platelets are recruited to the site of vas-
cular wall injury or ruptured atherosclerotic plaque through the
adhesive interactions between platelets and subendothelial matrix.
In response to collagen and soluble agonists such as thrombin and
adenosine diphosphate (ADP), platelets undergo activation, which
leads to formation of a stable thrombus via two simultaneous pro-
cesses. Activation of integrin αIIbβ3 (also known as GPIIb-IIIa)
allows formation of platelet-platelet homotypic aggregates via
fibrinogen molecular bridges forming a physical barrier at the site
of injury [1]. In response to strong stimulation with thrombin
and collagen, the rise in intracellular Ca++ triggers mitochondrial
collapse and scramblase activity on the plasma membrane in a sub-
set of activated platelets, termed procoagulant platelets. The
exposed phosphatidylserine (PS) on the surface of procoagulant
platelets supports formation of the prothrombinase complex and

Emmanuel J. Favaloro and Giuseppe Lippi (eds.), Hemostasis and Thrombosis: Methods and Protocols, Methods in Molecular
Biology, vol. 1646, DOI 10.1007/978-1-4939-7196-1_28, © Springer Science+Business Media LLC 2017

369
370 Leonardo Pasalic et al.

efficient generation of thrombin. The thrombin burst localizes

fibrin formation to the platelet aggregate, thereby stabilizing the
clot. Platelet aggregation and procoagulant activity combine to
form a thrombus [2, 3].
In the following chapter, we provide a number of protocols
aimed at assessment of constitutively expressed platelet membrane
receptors to diagnose inherited platelet bleeding disorders and
investigation of basal and agonist-induced platelet activation
responses including generation of platelet-leukocyte aggregates
(PLA), alpha and dense granule release, calcium flux, and PS
exposure.

2  Materials

2.1  Blood Collection 1. Evacuated sterile blood collection tubes containing 0.109 M
and Processing (3.2% w/v) sodium citrate (e.g., BD Vacutainer™, Franklin
Lakes, New Jersey; 363095).
2. Evacuated sterile blood collection tubes containing CTAD
(citrate, theophylline, adenosine, dipyridamole) (e.g., BD
Vacutainer™, Franklin Lakes, New Jersey; 367947). Ensure
that the tubes are protected from light at all times as light
exposure leads to degradation of the anticoagulants.
3. 21-gauge butterfly needles (e.g., Terumo, Leuven, Belgium;
SV*21BLK or Becton Dickinson, New Jersey, USA; 367246).
4. Sterile syringe 5 mL (e.g., Terumo, Laguna, Philippines; SS 05L).

2.2  Equipment 1. 96-well clear round bottom polypropylene microplate, not

treated, corner notch, individually wrapped, with lid, sterile
(e.g., Corning, Kennebunk, ME; 3879).
2. Adjustable (multichannel) pipettes.
3. 5 mL round bottom polystyrene flow tubes (optional snap
cap) (e.g., BD, 352054).
4. 0.22 μM Minisart syringe filters (e.g., Sartorius, 16534-K).
5. Flow cytometer (see Note 1).

2.3  Analysis 1. Dulbecco’s phosphate buffered saline (DPBS) (e.g., Gibco/

of Platelet Membrane Invitrogen).
Glycoproteins 2. 0.9% sodium chloride solution for irrigation (e.g., Baxter).
3. Formaldehyde (16%) aqueous solution, methanol free (e.g.,
Electron Microscopy Sciences, 15710) (see Note 2).
4. Fixative solution A (add 0.25 mL formaldehyde to 20 mL
0.9% sodium chloride solution). Filter before use.
5. Antibodies:
CD41a-PE (GpIIb; BD Biosciences, Clone HIP8, 555467).

2.4  Analysis
of Circulating Platelet-­
Leukocyte Aggregates
2.5  Analysis
of Platelet Dense
Granules
4.
2.6  Assessment
of Ex Vivo Platelet
Basal Activation
Flow Cytometry Protocols 371
CD42a-PE (GPIX; BD Biosciences, Clone ALMA.16,
558819).
CD42b-PE (GPIb; BD Biosciences, Clone HIP1, 555473).
CD61-PE (GPIIIa; BD Biosciences, Clone VI-PL2, 555754).
6. Isotype controls:
IgG PE Isotype Control (BD Biosciences, Clone MOPC-21;
554680).
7. Quantibrite beads—PE fluorescence quantitation kit (BD
Biosciences, 340495).
1. Dulbecco’s phosphate buffered saline (DPBS) (e.g., Gibco/
Invitrogen).
2. 10× Hank’s Balanced Salt Solution (HBSS) (e.g., Gibco/
Invitrogen).
3. Water for irrigation (e.g., Baxter).
4. Formaldehyde (16%) aqueous solution, methanol free (e.g.,
Electron Microscopy Sciences, 15710).
5. Antibodies and isotype controls:
CD42b-PE (BD Biosciences, Clone HIP1, 555473).
CD45-FITC (BD Biosciences, Clone HI30, 555482).
IgG PE Isotype Control (BD Biosciences, Clone MOPC-21,
554680).
6. Fixation solution B (0.315 mL formaldehyde, 0.3 mL 10×
Hank’s Buffered Saline Solution, 1.085 mL water).
7. Filter DPBS before use.
1. Dulbecco’s phosphate buffered saline (DPBS) (e.g., Gibco/
Invitrogen).
2. Formaldehyde (16%) aqueous solution, methanol free (e.g.,
Electron Microscopy Sciences, 15710).
3. Fixative solution A (see Subheading 2.3, item 4). Filter before use.
Mepacrine (quinacrine dihydrochloride) (Sigma Aldrich,
Q3251). Make up to a stock of 4 mM in PBS. Then further
dilute into a 30 μM working stock by adding 375 μL to 50 mL
of DPBS.
5. Antibodies
CD41a-PerCP-Cy5.5 (BD Biosciences, Clone HIP8, 340931).
1. Dulbecco’s phosphate buffered saline (DPBS) (e.g., Gibco/
Invitrogen).
2. 10× Hank’s Balanced Salt Solution (HBSS) (e.g., Gibco/
Invitrogen).
3. Water for irrigation (e.g., Baxter).
372 Leonardo Pasalic et al.

4. Fixative solution B: 0.16% formaldehyde prepared from form-

aldehyde (16%) aqueous solution (e.g., Electron Microscopy
Sciences, 15710) in 0.9% sodium chloride.
5. Antibodies and isotype controls:
CD42b-PE (BD Biosciences, Clone HIP1, 555473).
CD62P-PE (BD Biosciences, Clone AK-4 555524).
CD42b-FITC (BD Biosciences, Clone HIP1, 555472).
IgG1 PE (BD Biosciences, Clone MOPC-21, 555749).
PAC-1 FITC (BD Biosciences, Clone PAC-1, 340507).
IgM FITC (BD Biosciences, Clone G155-228 555583).

2.7  Assessment 1. Dulbecco’s phosphate buffered saline (DPBS) (e.g., Gibco/

of Agonist-Induced Invitrogen).
Platelet Activation 2. 0.9% sodium chloride solution for irrigation (e.g., Baxter).
3. Formaldehyde (16%) aqueous solution, methanol free (e.g.,
Electron Microscopy Sciences, 15710).
4. Fixative solution (A). Filter before use.
5. Antibodies and isotype controls:
C16.5D42b PE (BD Biosciences, Clone HIP1, 555473).
PAC-1 FITC (BD Biosciences, Clone PAC-1, 340507).
CD62P APC (BD Biosciences, AK-4, 550888).
CD63 PE-Cy7 (BD Biosciences, H5C6, 561982).
IgM FITC Isotype Control (BD Biosciences, G155-228,
555583).
IgG APC Isotype Control (BD Biosciences, MOPC-21, 555751).
IgG PE-Cy7 Isotype Control (BD Biosciences, MOPC-21,
557872).
6. Agonists (see Note 3).
(a) Thrombin receptor-activating peptide (SFLLRN) (e.g.,
Sigma Aldrich; S7152), made up to a stock concentration
of 400 μM in 0.9% sodium chloride (for testing at
0–80 μM).
(b) Adenosine diphosphate (ADP) (e.g., Sigma Aldrich, A2754).
(c)
Collagen-related peptide—cross-linked (CRP-XL)
(Department of Biochemistry, University of Cambridge),
final concentration 200 ng/mL.

2.8  Platelet 1. Phosphate-free Tyrode’s buffer (138 mM NaCl, 2.6 mM KCl,

Calcium Flux 12 mM NaHCO3, 5.5 mM glucose, 1.8 mM CaCl2, 0.49 mM
MgCl2, pH 7.4). Prewarmed to 37 °C.
2. Fluo-4 AM (Molecular Probes, F14201) reconstituted in
DMSO at 4 mM.
 Flow Cytometry Protocols 373

3. Platelet lineage antibody with a compatible fluorophore, e.g.,

CD42b PE (BD Biosciences, Clone HIP1, 555473).
4. Gly-Pro-Arg-Pro (GPRP) (Sigma Aldrich, G5779) made up to
40 mM in HBSS pH 7.35, stored in aliquots (15 μL, 30 μL) at
−20 °C.
5. Agonists as per experimental protocol.
(a) Thrombin from bovine plasma, 1000 U (e.g., Sigma
Aldrich, T4648-1KU) dissolved in water for injection at
1000 U/mL, stored in 10 μL aliquots at −80 °C.
●● 2 U/mL final concentration.
–– 1 in 25 dilution: 2 μL thrombin stock +48 μL
Tyrodes.
–– Add 25 μL to the assay for 2 U/mL final
concentration.
●● 0.5 U/mL final concentration.
–– Dilute further 1 in 4: 8 μL (1:25 dilution) + 24 μL
Tyrodes.
–– Add 25 μL to the assay for 0.5 U/mL reaction.
6. Microcentrifuge tubes (0.6 and 1.5 mL).
7. Protein gel loading tips (e.g., BIO-RAD, 223-9915).

2.9  Procoagulant 1. Hank’s Balanced Salt Solution (HBSS), no calcium, no mag-

(PS-Exposing) nesium, and no phenol red (e.g., Gibco, 14175095). pH
Platelets adjusted to 7.35.
2. Wash buffer (HBSS with 0.35% human serum albumin,
pH 7.35).
3. GPRP (Sigma Aldrich, G5779) made up to 40 mM in HBSS
pH 7.35, stored in aliquots (15 μL, 30 μL) at −20 °C.
4. CaCl2 solution in water (100 mM).
5. Agonists as per experimental protocol, e.g.:
Thrombin from bovine plasma, 1000 U (e.g., Sigma Aldrich,
T4648-1KU) dissolved in water for injection at 1000 U/
mL, stored in 10 μL aliquots at −80 °C.
Collagen, e.g., equine tendon collagen type I (Collagen
Reagens Horm, Takeda, Austria).
6. Antibodies and isotype controls:
Lactadherin conjugated to FITC (Haematologic Technologies,
BLAC-FITC).
CD41a BV510 (BD Biosciences, Clone HIP8, 563250).
CD45 BUV395 (BD Biosciences, Clone HI30, 563792).
CD62P PE (eBioscience, Clone Psel.KO2.3, 12-0626-82).
IgG1 K Isotype Control PE (eBioscience, 12-4714-42).
7. Fixative PamFix (Platelet Solutions, PSR-001) stored at 4 °C.
374 Leonardo Pasalic et al.

3  Methods

3.1  Blood Collection 1. General method.

(a) Obtain whole blood by standard venipuncture using a
21G butterfly needle with no or minimal stasis into a
citrate tube.
(b) Discard the first tube collected.
(c) Ensure the tube is completely filled and mix by gentle
inversion 4–5 times.
(d) The tube(s) should be transported in a vertical position to
the laboratory at room temperature within 15 min of col-
lection to minimize artificial platelet activation.
2. Method for assessment of basal level of activation (see
Subheading 3.5).
(a) Using 2 × 5 mL syringes, collect blood into a 21-G but-
terfly cannula with the tourniquet removed after insertion
of the needle.
(b) Discard the first 3 mL of blood.
(c) Collect a further 5 mL of blood into a fresh syringe at a
rate of approximately 0.5 mL/min (low suction) to pre-
vent spurious platelet activation.
(d) Uncap the CTAD tube and fill with blood to the fill line
manually to further reduce activation.
(e) Mix the blood by gentle inversion 4–5 times and store at
4 °C during transport and until assayed.

3.2  Platelet 1. Set up and label flow tubes before blood collection as per
Membrane Table 1.
Glycoproteins 2. Add appropriate antibodies to the tubes (see Table 1).
3. Add 5 μL of citrated whole blood to each tube.
4. Mix by gently shaking the tube. Do not vortex.

Table 1
Tube setup for platelet glycoprotein assay

Test tube (one

antibody per
Reagent Control tube (PE isotype) tube)
PE-conjugated antibody (μL)  5
PE isotype control (μL)  2.5
DPBS (μL) 42.5 40
PE phycoerythrin, DPBS Dulbecco’s phosphate buffered saline
 Flow Cytometry Protocols 375

Table 2
Tube setup for circulating platelet-leukocyte aggregate assay

Platelet-­leukocyte
Reagent PE isotype control aggregates
CD45-FITC (μL)  5  5
PE isotype control (μL)  2.5
CD42b-PE (μL)  5
DPBS (μL) 67.5 65
FITC fluorescein isothiocyanate, PE phycoerythrin, DPBS Dulbecco’s phosphate buff-
ered saline

Table 3
Tube setup for assay of platelet dense granules

Reagent Control tube Test tube

CD41-PerCP-Cy5.5 (μL)  5  5
Mepacrine (μL) 20
DPBS (μL) 20
PerCP peridinin-chlorophyll proteins, Cy5.5 cyanine 5.5, DPBS Dulbecco’s phosphate
buffered saline

5. Incubate for 20 min at room temperature.

6. Add 0.5–1.0 mL of fixative solution A to each tube.
7. Incubate for 10 min.
8. Analyze on flow cytometer within 6 h.

3.3  Analysis
of Circulating Platelet-­
Leukocyte Aggregates
1. Set up and label flow tubes before blood collection.
2. Add appropriate antibodies to the tubes (see Table 2).
3. Add 25 μL of citrated whole blood to each tube (see Note 4).
4. Mix by gently shaking the tube. Do not vortex.
5. Incubate for 10 min at room temperature.
6. Add 84 μL of fixative solution B.
7. Incubate for 10 min in the dark at room temperature.
8. Add 840 μL of H2O and incubate for 10–15 min (see Note 5).
9. Analyze on flow cytometer within 2 h.

3.4  Analysis 1. Prior to blood collection, set up and label tubes (see Table 3).
of Platelet Dense 2. Add whole blood to the tubes (5 μL/tube), and mix by gently
Granules flicking.
376 Leonardo Pasalic et al.

Table 4
Tube setup for assessment of ex vivo platelet basal activation levels

Tube no. Antibody 1 Antibody 2 DPBS

1 CD42b FITC, 5 μL IgG PE 5 μL 35 μL
2 CD42b FITC 5 μL CD62P PE 5 μL 35 μL
3 IgM FITC 5 μL CD42b PE 5 μL 35 μL
4 PAC-1 FITC 5 μL CD42b PE 5 μL 35 μL
FITC fluorescein isothiocyanate, PE phycoerythrin, DPBS Dulbecco’s phosphate
buffered saline

3. Incubate at room temperature in the dark for 20 min.

4. Add 1 mL of fixative solution A per tube.
5. Incubate for 10 min.
6. Centrifuge tube at 1500 × g for 5 min.
7. Carefully remove the top 900 μL of supernatant.
8. Add 900 μL of DPBS.
9. Analyze on flow cytometer within 2 h.

3.5  Assessment 1. Prior to blood collection, set up and label flow tubes (see
of Ex Vivo Platelet Table 4).
Basal Activation 2. Add whole blood from CTAD tube to the prepared tubes
(5 μL/tube), and mix gently by flicking.
3. Incubate at 4 °C in the dark for 20 min.
4. Add 1 mL of ice-cold fixative solution C (see Note 6) per tube.
5. Analyze on flow cytometer within 2 h of fixation.

3.6  Assessment 1. Prior to blood collection, set up tubes according to Table 5.

of Agonist-Induced 2. Add whole blood from citrate tube and mix gently.
Platelet Activation
3. Incubate at room temperature in the dark for 20 min.
4. Add 1 mL of fixative solution A per tube.
5. Analyze on flow cytometer within 2 h of fixation.

3.7  Platelet 1. Add 50 μL of citrated whole blood to 1.5 mL microcentrifuge

Calcium Flux tube.
2. Add 0.625 μL of 4 mM Fluo-4 AM and 450 μL Tyrode’s buffer
(37 °C) to the tube. Mix well (see Note 7).
3. Incubate in water bath at 37 °C for 15 min.
4. While Fluo-4 AM is being loaded, prepare 1.5 mL microcen-
trifuge tubes for staining. One tube is sufficient for one agonist
in duplicate.
 Flow Cytometry Protocols 377

Table 5
Tube setup for agonist-induced platelet activation assay

Reagent Control tube (μL) Test tube (μL)

CD42b-PE 5 5
IgM FITC 5
IgG APC 5
IgG PE-Cy7 2.5
PAC-1 FITC 5
CD62P APC 5
CD63 PE-Cy7 2.5
Agonist (if required) 10 10
DPBS Total volume made up 50 μL
PE phycoerythrin, FITC fluorescein isothiocyanate, APC allophycocyanin, Cy7 cyanine
7, DPBS Dulbecco’s phosphate buffered saline

5. To each tube add:

1 μL CD42b PE for platelet labeling.
1.75 μL of 40 mM GPRP (final concentration 2.5 mM).
6. Transfer 25 μL of Fluo-4 AM loaded whole blood to above
tubes.
7. Incubate at 37 °C for 15 min.
8. Dilute with 1 mL of Tyrode’s buffer (37 °C).
9. Split stained sample volume to 2 × 500 μL in 0.6 mL micro-
centrifuge tubes for acquisition.

3.8  Procoagulant 1. Prepare the reaction buffer (HBSS without calcium, magne-
(PS-Exposing) sium, and phenol red, containing 2.5 mM GPRP, 2.5 mM
Platelets CaCl2) (see Table 6).
2. Prepare the stain mix according to Table 7.
3. Prepare working dilutions of agonists in HBSS and keep on ice
until use, e.g.:
Thrombin (final concentration 2 U/mL).
Collagen (final concentration 10 μg/mL).
4. Label FACS tubes and dispense 3 mL of the wash buffer into
each FACS tube.
5. To prepare 96-well reaction plate (see Note 8):
(a) Dispense 30 μL of reaction buffer to each reaction well
(column 2) just before blood collection (see Note 9).
(b) Add agonist or vehicle control (HBSS) in 5 μL to each
reaction well (column 2).
378 Leonardo Pasalic et al.

Table 6
Reaction buffer composition

Reagent n (wells) Volume (μL)

HBSS 1 24.7
GPRP (40 mM) 1  3.1
CaCl2 (100 mM) 1  2.2
Agonist or substitute with HBSS 1 5
(Final reaction volume after addition of 15 μL whole blood is 50 μL)
HBSS Hank’s Balanced Salt Solution, GPRP Gly-Pro-Arg-Pro

Table 7
Stain cocktail

Fluorescence minus Platelets and platelet-­

Reagent PE isotype control FITC control leukocyte aggregates
CD41a-BV510 (μL) 2.8 2.8 2.8
CD45-BUV395 (μL) 2.5 2.5 2.5
Lactadherin-FITC (μL) 3.125 3.125
PE isotype control (μL) 0.625 0.625
CD62P-PE (μL) 0.625
HBSS (μL) 70.95 74.075 70.95
BV510 Brilliant Violet 510, BUV395 Brilliant Ultraviolet 395, FITC fluorescein isothiocyanate, PE phycoerythrin,
HBSS Hank’s Balanced Salt Solution

(c) Add 170 μL of HBSS to wells adjacent to reaction wells

(column 3) (see Note 10).
(d) Dispense 50 μL of citrated whole blood gently without
introducing bubbles into column 1 wells (see Note 11).
6. Transfer 15 μL of citrated whole blood to each reaction well
(column 2) using multichannel pipette and gently mix (if using
electronic pipettor set mix volume to 30 μL, moderate speed).
7. Incubate for 10 min at room temperature.
8. Dispense stain mix to stain wells (column 4) (80 μL/well).
Place in the dark at room temperature.
9. At the end of the 10 min incubation period, transfer HBSS
(150 μL) (from column 3) to each reaction well (column 2) and
mix gently (see Note 12). Transfer 20 μL of diluted stimulated
whole blood to the wells containing the stain mix (column 4).
 Flow Cytometry Protocols 379

Transfer another 20 μL from a dual agonist-stimulated well to

the well containing control stain. Mix gently.
10. Incubate for 15 min in the dark at room temperature.
11. During staining, dispense 220 μL of PamFix to unoccupied
wells (column 5).
12. Add 200  μL of PamFix to all stain and control wells and mix
gently.
13. Incubate for 5 min in the dark at room temperature.
14. After gentle mix, transfer the fixed and stained cell suspension
into the FACS tubes containing 3 mL of wash buffer.
15. Centrifuge at 1500 × g for 8 min (medium acceleration/
deceleration).
16. Decant the supernatant.
17. Resuspend the pellet in the residual fluid by giving the FACS
tube a few gentle flicks.
18. Add 1 mL of wash buffer to the FACS tubes.

4  Flow Cytometric Acquisition and Analysis

4.1  Platelet 1. Set threshold to a low value on forward scatter (FSC).

Membrane 2. On a FSC vs side scatter (SSC) or FSC vs fluorescence plot
Glycoproteins (axes must be set to a log scale or biexponential scale), adjust
the voltages to locate the platelet population (see Note 13).
3. Acquire at least 10,000 platelet events for analysis (Fig. 1).
4. Assess the mean fluorescence intensity for each platelet
antibody.
5. Quantibrite beads can be used to convert the mean fluores-
cence intensity into the number of PE molecules bound to the
platelet.
(a) Add 500 μL of DPBS to a Quantibrite tube.
(b) Analyze on the flow cytometer to determine the mean
fluorescence intensity of the four bead populations (low,
med-­low, med-high, and high).
(c) Generate a standard curve according to the supplied bead
intensities.

4.2  Circulating 1. Ensure the cytometer flow rate is set to a low speed/flow rate
Platelet-Leukocyte (see Note 14).
Aggregates 2. Set the threshold to fluorescence in the FITC channel to
acquire only the CD45 positive population (see Note 15).
3. On a FSC vs SSC plot (axes set to linear scale), three different
leukocyte subpopulations can be identified on the basis of
380 Leonardo Pasalic et al.

a b
5
10 105

104 104
FSC-A

FSC-A
103 Platelet 103
Platelet
gate gate
102 102
0 0

0 102 103 104 105 0 102 103 104 105

SSC-A CD42b
c d
100 100

80 80
% of max

% of max

60 60

40 40

20 20

0 0
0 102 103 104 105 0 102 103 104 105
CD42a CD42b
e
100

80
% of max

60

40

20

0
0 102 103 104 105
CD41a
Fig. 1 Assessment of platelet glycoprotein receptor expression. The platelet populations can be identified in a
normal donor on the light scatter characteristics (FSC-A vs SSC-A or FSC-H vs SSC-H) (a) or CD42b vs FSC-A
(b). Using a histogram of this platelet population, the expression of CD42a (c), CD42b (d), or CD41a (e) can be
determined. Background levels are established for platelets (black), and in this example the expression of each
marker is shown for a normal donor (blue), or a patient with Bernard-Soulier syndrome (red). Note the
decreased expression of CD42a and CD42b on the patient’s platelets, but the increased expression of CD41a
 Flow Cytometry Protocols 381

their light scatter profile alone or in combination with CD45

expression: granulocytes, monocytes, and lymphocytes
(Fig. 2).
4. Acquire at least 1000 monocyte events for analysis (see Note 16).
5. Using the data acquired on a sample dual-labeled with CD45-­
FITC and with the isotype control for the platelet marker (PE),
set the boundary for the PE positive events (PLA) for each of
the three leukocyte subpopulations based on capturing 0.5% of
the isotype control.
6. Run the samples labeled for both CD45-FITC and CD42b-PE
and analyze the percentage of each of the leukocyte popula-
tions that falls into the PLA region (Fig. 2).

4.3  Assessment 1. Samples are analyzed on the flow cytometer on “low” flow rate
of Platelet Dense (see Note 14).
Granules 2. Identify the platelets by a combination of FSC and SSC prop-
erties and CD41a expression.
3. Assess platelet mepacrine staining using either a blue (488 nm)
or violet (405 nm) laser, with detector at 525 nm (similar to
FITC fluorescence).
4. Adjust a marker gate such that at background it corresponds to
0.5% staining in the control tube. Alternatively, mean fluores-
cence intensity can be used as the assay output.
5. Acquire at least 10,000 platelet events for analysis.

4.4  Assessment 1. Samples are analyzed on the flow cytometer on “low” flow rate
of Ex Vivo Platelet (see Note 14).
Basal or Agonist-­ 2. Identify the platelet cloud by a combination of FSC and SCC
Induced Activation properties and CD42b expression (a marker/gate set such that
at background it corresponds to 0.5% expression in the isotype
control tube) (Fig. 3b). Alternatively, mean fluorescence inten-
sity can be used (geometric mean for all fluorophores) to assess
the change in the population as a whole rather than looking
only at the percentage of positive cells (Fig. 3c–e).
3. Acquire at least 10,000 platelet events for analysis.

4.5  Platelet Calcium 1. Before placing tube on BD Accuri™ (see Note 17) for collec-
Flux tion, attach “protein gel loading tip” to 200 μL pipette and
load with 25 μL of agonist working dilution.
2. With the agonist still in the pipette tip, slowly adjust the load-
ing volume to approximately 125 μL (the additional 100 μL
volume is to allow mixing after the addition of the agonist to
the blood sample that is on the SIP of the BD Accuri™).
3. Place tube on BD Accuri™.
 a b
1.0K 1.0K

Granulocytes (CD45+)
800 Granulocytes 800

600 600
SSC-H

SSC-H
400 400
Monocytes (CD45+)
Monocytes
200 Lymphocytes
200

Lymphocytes (CD45+)
0 0
0 200 400 600 800 1.0K 100 101 102 103 104
FSC-H CD45 FITC
c d
Isotype Control 150
Platelet-
120 (granulocytes) Granulocyte
Aggregates
90 100
Counts

Counts

60 0.53% 2.39%
50
30

0 0
100 101 102 103 104 100 101 102 103 104
IgG PE CD42b PE
e f
25
Isotype Control
15
20
(monocytes) Platelet-
Monocyte
15 Aggregates
Counts

Counts

10

10 0.55% 19.6%
5.0
5.0

0 0
100 101 102 103 104 100 101 102 103 104
IgG PE CD42b PE

Fig. 2 Assessment of platelet-leukocyte aggregates. The leukocyte populations can be identified either on the
light scatter characteristics (FSC-H vs SSC-H) (a) or CD45 vs SSC-H (b). Using a histogram, background levels
(0.5%) are established for each cell type, i.e., select a leukocyte subpopulation and set background levels on
the isotype control (c and e). Once the background is established, the percentage of platelet-granulocyte/
monocyte/lymphocyte aggregates can be determined (d and f)
 Flow Cytometry Protocols 383

a b
Ungated Baseline
104 104
Q1 Q2
0 0.85

103 103
Platelet
gate

CD62P
SSC-H

102 102

101 101
Q4 Q3
0 99.2
100 100
100 101 102 103 104 100 101 102 103 104
CD42b CD42b
c Agonist treated
d Agonist treated
100 100

80 80
CD63+
% of max

% of max

60 60 PAC1+

40 40

20 20

0 0
0 102 103 104 105 0 102 103 104 105
CD63 PAC1
e Agonist treated
100

80

CD62P+
% of max

60

40

20

0
0 102 103 104 105
CD62P APC

Fig. 3 Evaluation of platelet activation. Platelets (defined by SSC-H and CD42b) (a) were collected in CTAD to
estimate level of activation at baseline (b) or collected in citrate and stimulated with agonists: SFLLRN (red),
CRP-XL (green), and ADP (blue) (c–e)
384 Leonardo Pasalic et al.

4. Set acquisition threshold based on the fluorescence of the

platelet lineage marker. Threshold must be established for indi-
vidual cytometer, antibody, and conjugate.
5. Collect for at least 20 s at slow flow rate to establish the
baseline.
6. Add agonist and mix immediately, pipetting up and down six
times.
7. Run at slow speed for a total of 5 min (see Note 18).
8. Analysis should be performed on a platelet gate defined by
FSC and fluorescent platelet marker. Samples can be analyzed
according to the percentage of platelets with Fluo-4 signal
above baseline over time or median Fluo-4 fluorescence inten-
sity over time using a flow cytometry software package with a
kinetics option (Fig. 4).

4.6  Procoagulant 1. Set threshold to a low value on forward scatter (FSC).

(PS-Exposing) 2. Gate on platelets defined as (CD41a+/CD45-) (Fig. 5).
Platelets
(a) Biexponential scaling is helpful for visualizing all events in
a heterogeneous complex cellular matrix like whole blood.
3. Collect at least 10,000 platelet events for analysis.
4. Aim for event rate of around 2000 total events per second to
minimize coincident events. This rate will depend on a particu-
lar flow cytometer.
5. Analyze the events from the platelet gate on a plot of CD62P
PE vs lactadherin FITC fluorescence.
(a) Use the isotype PE control sample to set the threshold for
CD62P positive events.
(b) Use the fluorescence minus FITC control to set the thresh-
old for lactadherin positive events (Fig. 5) (see Note 19).

5  Notes

1. The minimum/optimum flow cytometer configuration (num-

ber of lasers and detection photomultiplier tubes) will depend
on the complexity of each individual panel/experiment. Older
instruments with a single excitation laser (488 nm) are usually

Fig. 4 (continued) expression and then analyzed for change in intracellular calcium concentration over time.
Results are shown for the gating strategy (a and c) and change in calcium levels over time after vehicle control
(b) and after thrombin 2 U/mL agonist stimulation (d). Analysis using kinetics function by FCS Express™ is
shown according to the percentage of platelet events with intracellular calcium levels above threshold (e) and
median Fluo-4 AM fluorescence over time (f)
 Flow Cytometry Protocols 385

a b
Vehicle (ungated) Vehicle (platelet gate)
107 107
d
106 h ol
106
r es
105 Th
105

Fluo-4 AM
SSC-A

104

Platelet 104
103
gate
102 103
101 -102
100 -103
100 101 102 103 104 105 106 107 80 160 240 320
CD42b PE Time (Sec)
c d
Thrombin (ungated) Thrombin (platelet gate)
107 107
106 o ld
106 sh
re
105 Th
Fluo-4 AM

105
SSC-A

104
Platelet 104
103
gate
102 103

101 102

100 -102
100 101 102 103 104 105 106 107 80 160 240 320
CD42b PE Time (Sec)
e f
Thrombin (platelet gate) Thrombin (platelet gate)
100
105
% Above Threshold

Median Fluo-4 AM

75

50

25
104

80 160 240 320 80 160 240 320

Time (Sec) Time (Sec)
Fig. 4 Measuring platelet calcium flux in whole blood. Whole blood was incubated with calcium-sensitive dye
Fluo-4 AM and platelet marker anti-CD42b-PE. Platelets are identified by SSC characteristics and CD42b
386 Leonardo Pasalic et al.

Isotype PE minus
a Thrombin/collagen b FITC (platelet gate)

105 Q1 Q2 105 Q1 Q2
1.72 0.83 0.51 0.77
CD45-BUV 395

IgG1-PE
104 104

Platelet gate
103 103

102
0 Q4 Q3 0 Q4 Q3
43.1 54.3 -102 98.1 0.62

0 103 104 105 0 103 104 105

CD41a BV510 FITC

c Thrombin/collagen
(platelet gate)

105 Q1 Q2
38.5 60.0
CD62P PE

104

103

102
0 Q4 Q3
-102 0.50 1.01

0 103 104 105

Lactadherin-FITC

Fig. 5 Example of flow cytometric analysis phosphatidylserine exposure on platelet membrane. Whole blood
samples were incubated with CD45 BUV395, CD41a BV510, CD62P PE, and lactadherin FITC or controls. (a)
Platelets are identified in lower right quadrant “platelet gate” as CD41a+ve/CD45−ve. Thresholds for CD62P
and lactadherin are set by isotype control (PE) and fluorescence minus FITC control at 0.5% positivity (b).
Platelets with alpha-granule release and PS exposure are identified by CD62P+ve/lactadherin+ve (top right
quadrant)

adequate for a 2–3 color experiment, but the setup can be

more complicated due to compensation issues. Collecting
uncompensated data and performing compensation using a
software package can simplify this process. Newer instruments
equipped with at least three lasers (violet, 405 nm; blue,
488 nm; and red, 631 nm) provide increased flexibility with
respect to the number of parameters measured often with no
or minimal spillover.
 Flow Cytometry Protocols 387

2. The terms formaldehyde, paraformaldehyde, and formalin all

refer to formaldehyde (CH2O), which is the simplest aldehyde.
Formalin is the name commonly used for saturated (37%)
formaldehyde solution [4]. Therefore, a protocol stating the
use of 1% formalin would approximately be equivalent to 0.4%
formaldehyde. Of note, some formalin solutions contain meth-
anol to prevent polymerization, and it may have negative effect
on the analyte in question. Paraformaldehyde (PFA) is polym-
erized formaldehyde [HO(CH2O)nH] in solid form. However,
“pure,” methanol-free formaldehyde solution is referred to by
some authors as PFA, although it does not contain the poly-
mer forms.
3. If there is a desire to maintain consistency across different
whole blood platforms (flow cytometry, Multiplate™ imped-
ance aggregometry) within a single study, consideration should
be given to using agonists provided by a single supplier. Both
ADP and TRAP used in the Multiplate™ assay have been used
by one of the authors (DC) to stimulate platelet activation.
4. For assessment of basal level PLA in circulation, collect blood
into CTAD-containing tubes as per Subheading 3.1, step 2.
5. This ensures complete red blood cell lysis, which assists in
identification of platelets during flow cytometric analysis.
6. Filter the 0.16% formaldehyde platelet fixative solution with a
0.22  μm syringe filter (Millipore) to remove any particulates
that may be present.
7. To prevent possible clotting of the blood due to calcium in the
Tyrode’s buffer, the GPRP may be added in the initial incuba-
tion step instead of with the platelet antibody.
8. An experiment template for the 96-well plate indicating loca-
tion of reaction wells, wells containing HBSS, stain cocktail,
etc., can be very useful in ensuring all steps are easy to execute.
See Fig. 6 for an example.
9. Allow all reagents within the plate wells to come to room tem-
perature prior to adding whole blood.
10. This will be used to transfer HBSS to the reaction wells to
dilute the agonist and Ca++ in the reaction wells after 10 min
incubation period with whole blood.
11. Do not attempt to expel the contents of the pipette tip beyond
the first stop to avoid introducing bubbles. Dispense blood
within 10 min of collection.
12. Cells tend to settle a bit by the end of 10 min, so direct the
pipette tip to several different directions to produce even
suspension.
13. As platelets are smaller than erythrocytes and leukocytes, the
forward scatter and side scatter voltages are typically set
388 Leonardo Pasalic et al.

Fig. 6 Layout of the 96-well plate for procoagulant (PS-exposing) platelet assay.
The plate is set up as shown. The reaction wells should be prepared just prior to
blood collection. All reactions, staining and fixing, must be performed at room
temperature

higher than voltages for these cell types. This will move the
erythrocytes to the end of the axis.
14. In order to minimize coincidence events, the flow cytometer
should be run at a low speed/flow rate. At higher speeds the
probability increases that unbound platelets and leukocytes
will be detected as a single event.
15. In order to select the appropriate fluorescence threshold, first
plot CD45 fluorescence (x-axis) vs forward scatter (y-axis),
with the threshold on forward scatter that is suitably low to
identify the lymphocyte population. Then change the thresh-
old to fluorescence and gradually increase until CD45-­negative
events are no longer detected.
16. In a normal individual, acquiring 1000 monocyte events
should allow for sufficient granulocytes and lymphocytes to be
collected; however, these may vary in patients with different
pathologies. Ensure that 1000 of each population is collected
as a minimum.
17. This method utilizes the Accuri C6 BD which operates via a
peristaltic pump action, permitting use of open tubes. This is
an advantage in calcium flux as it allows addition of agonist
without interruption of data collection. However, calcium flux
can also be performed on flow cytometers with pressurized,
closed fluidic systems with acceptance that a gap will inevitably
appear in the data collection. If using pressurized system, con-
sider preparing samples in duplicate so that while running the
baseline the agonist can be added and used to replace the base-
line sample on the SIP quickly.
 Flow Cytometry Protocols 389

18. After each agonist, backflush the sample line, and then run BD
Accuri™ cleaning solution for 30 s, followed by 30 s of Milli-­Q
H2O to remove residual protein. This is now ready for the next
sample.
19. Use isotype PE rather than CD62P-PE for this control to
avoid PE spillover to the FITC channel.

References

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2. Sangkuhl K, Shuldiner AR, Klein TE, Altman
RB (2011) Platelet aggregation pathway.
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3. Ferroni P, Vazzana N, Riondino S, Cuccurullo
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