Supplementary Information

Evaluation of Novel Prostate-Specific Membrane Antigen-Targeted Near Infrared Imaging

Agent for Fluorescence-Guided Surgery of Prostate Cancer

Sumith A. Kularatne,1, * Mini Thomas,1 Carrie H. Myers,1 Pravin Gagare,1 Ananda K.

Kanduluru,1 Christa J. Crian2, Brandy N. Cichocki2

1 On Target Laboratories, 1281 Win Hentschel Blvd, West Lafayette, IN, 47906 USA

2 Department of Veterinary Clinical Sciences, Purdue University, Lynn Hall, 625 Harrison St.,

West Lafayette, IN 47907 USA

* correspond: Sumith A. Kularatne, Ph.D. On Target Laboratories, 1281 Win Hentschel Blvd,

West Lafayette, IN, 47906, Tele: 765-558-4547, Fax: 765-598-4452, email:

skularatne@ontargetlabs.com

1

SI Figures, Tables, and Schemes

SI Figure 1: (a) Chemical structures of S0456 and DUPA-FITC. (b) Excitation (Ex) & emission

(Em) spectra of OTL78 (1 μM) andS0456 (1 μM) in 1 mL of PBS obtained using fluorometer.

(c) Evaluation of PSMA expression levels in LNCaP, 22Rv1, PC3, and A549 using flow

cytometry. (d) Dose dependent binding of DUPA-FITC and (e) competitive binding of OTL78

with respect to DUPA-FITC to 22Rv1 and PC3 cells in culture. Error bars represent SD (n = 2).

(f)Binding and internalization of OTL78 to (i) 22Rv1 and (ii) LNCaP at 4 ºC by epifluorescence

microscopy. Nuclear is stained with DAPI (a blue dye).

2

SI Figure 2: NMRs of OTL78. (a) 1H – NMR and (b) 13C – NMR of OTL78 using Brooker 500

and 125 MHz spectrometer.

3

SI Figure 3: Mass spectra of OTL78. (a) HPLC chromatogram using Waters UPLC system and

(b) MS using UPLC coupled with MS.

4

SI Figure 4. Tissue biodistribution analysis of OTL78: (a) IVIS images showing overlay of

fluorescence images over white light images of selected tissues and (b) tumor-to-tissue ratio

from tissue biodistribution data from mice bearing 22Rv1 tumor xenografts after administering

increasing doses of OTL78. Error bars represents SD (n= 5).

5

SI Figure 5: In vivo efficacy of OTL78. Tissue biodistribution analysis using fluorescence

imaging of the mice with (a) PC3 and (b) A549, and (h) 22Rv1 orthotopic tumors at 2h post-

injection. Representative fluorescence images from AMI imager showing mice bearing (c)

22Rv1 orthotopic (n=5 mice/group) and (d) tissue biodistribution analysis using fluorescence

imaging of the same mice 2h after administering 10 nmol of OTL78.

6

SI Figure 6. Quantitation of TBR of OTL78 using region of interest (ROI) and ImageJ analysis.

(a) TBR calculated using ROI values obtained from IVIS or AMI imager after tissue

biodistribution studies of 22Rv1 subcutaneous or orthotopic tumors bearing mice injected with

10 nmol of OTL78. Note: Since the primary tumor is in the prostate, tumor-to-prostate ratio is

equal to one in orthotopic model. Error bars represents SD (n=5 mice/group). (b) Representative

fluorescence image (in gray scale) of mouse bearing 22Rv1 subcutaneous tumor after injecting

10 nmol of OTL78. The plot of gray value versus distance (c) across the line and (d) within the

box are shown in the Fig 4b.

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SI Figure 7. Comparison of surgeries performed under conventional and fluorescence-guided

techniques. (a) Representative fluorescence images of mice before and after surgically removing

22Rv1 tumor xenografts by conventional (n=5 mice/group) or fluorescence-guided (n=5

mice/group) techniques till day 21. Mice were administered with OTL78 (10 nmol/mouse) 2 h

before imaging using AMI image system. The cohort underwent on fluorescence-guided surgery

were monitored over a 30 days.

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SI Figure 8: Time dependent tumor retention of OTL78. IVIS images showing time dependent

whole-body fluorescence images over white light images of mice bearing 22Rv1 tumor after

injecting 10 nmol of OTL78 and image at different time intervals (n=5 mice/group).

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SI Figure 9: Human serum binding studies of OTL78. Serum binding is defined as the ratio of

area under the OTL78 peak in serum (sample): area under the OTL78 peak in PBS (negative

control). n=3.

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SI Figure 10: Safety of OTL78. Histopathological analysis mice treated with OTL78 (10

µmol/mouse). a: Cerebellum, b: Cerebrum, c: Heart, d: Kidney, e: Large intestine, f: Liver, g:

Lung, h: Skin, i: Muscle, j: Spleen, k: Stomach, l: Small intestine (n=5 mice/group).

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SI Tables

SI Table 1: Percent of basophile activation in healthy subjects

Note: fMLP = N-formylmethionyl-leucyl-phenylalanine is a non-specific cell activator, anti-

FcεR = a high affinity monoclonal antibody binding to IgE, and stimulation index (SI) is defined

as the ratio of %basophil activation by the allergen: %basophil activation by the background

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SI Schemes

SI Scheme 1: Reagents and conditions for synthesis of OTL78: (a) (i) 2: H2N-Tyr(OtBu)-

OtBu.HCl, HATU, DIPEA, DMSO, RT, 2h; (b) H2/ Pd-C (10%), DCM/MeOH; (c) 5: Cbz-PEG2-

CO2H, HATU, DIPEA, DMSO, RT, 2h; (d) H2/ Pd-C, DCM/MeOH; (e) 7, HATU, DIPEA,

DMSO, RT, 2h; (f) TFA, RT, 1h; (g) (i) H2O/ NaOH (pH = 10), (ii) 11: S0456, H2O, 75 °C, 3-

4h.

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SI Materials & Methods

Materials: All the amino acids and O-t-Butyl-L-tyrosine t-butyl ester hydrochloride and O-(7-

azabenzo-triazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU) were

obtained from Chem-Impex Int (Chicago, IL). DUPA was custom made at Laviana Pharmatech

Co. (Beijing, China) and CbzNH-PEG2-COOH was purchased from Santa Cruz Biotechnology

(Dallas, TX). S0456 is custom made at Eastman Kodak Company (Rochester, NY). All other

chemicals, cell culture materials, and animal supplies were obtained from major suppliers.

General methods: Moisture and oxygen sensitive reactions were carried out under an argon

atmosphere. OTL38 was purified by preparative reverse phase (RP)-HPLC (Waters, xTerra C18

10 μm; 19 x 250 mm) and analyzed UPLC (Acquity, BEH C18 1.7 μm; 2.1 x 50 mm) using

solvent as A = 10 mM NH4OAc (pH = 7.0) and B = CH3CN. 1H- & 13C spectra were acquired

with a Bruker 500 MHz and 125 MHz NMR spectrometer equipped with a TXI cryoprobe.

Samples were run in 5mm NMR tubes using D2O. Pre-saturation was used to reduce the intensity

of the residual H2O peak. All 1H signals are recorded in ppm with reference to residual DMSO

(2.50 ppm) and data are reported as s = singlet, d = doublet, t = triplet, q = quartet, and m =

multiplet or unresolved, b = broad, with coupling constants in Hz. LC/MS analyses were

obtained using a Waters micromass ZQ 4000 mass spectrometer coupled with a UV diode array

detector. High resolution mass spectrometry (HRMS) results were obtained by Electron

Ionization Spray (ESI) using an Applied Biosystems (Framingham, MA). Animal imaging

experiments were performed using a Caliper IVIS Lumina II Imaging Station with Living Image

4.0 software (PerkinElmer Inc, MA) with image parameters of ex= 745 nm, em = ICG, and

exposure time = 1s or using Ami HT Image station with AMIView Image Analysis Software

with image parameters of ex= 745 nm, em = 810 nm, and exposure time = 1s.

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Cell culture: LNCaP, 22Rv1 and PC3 (human prostate cancer cell lines) and A549 (a alveolar

basal epithelial carcinoma cell line) cells were obtained from American Type Culture Collection

(ATCC) (Rockville, MD, 2014) and grown as a monolayer using normal 1640 RPMI-medium

(Gibco, NY) containing 10% heat-inactivated fetal bovine serum (Atlanta Biological, GA) and

1% penicillin streptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidified atmosphere

at 37 ˚C for at least 4 passages before they were used for the assays.

Animal: Athymic male nude (nu/nu) (7 weeks old, 20-25 g) were purchased from Invigo

(Indianapolis, IN) and maintained on normal diet. Animals were housed 5/cage in a barrier,

pathogen-free cloaked rack. Autoclaved tap water and food were given as needed. The animals

were housed in a sterile environment on a standard 12 h light-dark cycle for the duration of the

study. All animal procedures were approved by Purdue Animal Care and Use Committee.

Animal care and studies were performed according to national and international guidelines for

the humane treatment of animals.

Animal imaging experiments were performed using a Caliper IVIS Lumina II Imaging Station

with Living Image 4.0 software (PerkinElmer Inc, MA) using imager parameters as ex= 745 nm,

em = ICG, and exposure time = 1s. ROI calculations were conducted using Living Image 4.0

software.

Human blood: Collection of blood samples from human subjects and further studies were done

according to a Purdue University approved Institutional Review Board protocol.

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Synthetic Procedures: Synthesis of OTL78

Synthesis of CbzNH-Phe-Tyr-(OtBu)-OtBu (3): A 2000 mL round bottom (rb) flask was

charged with a stir bar, (L)-CbzNH-Phe-OH [(1), 50.0 g, 167.04 mmol, 1.0 equiv.)], (L)-NH2-

Tyr(-OtBu)-OtBu· HCl [(2), 155.10 g, 167.04 mmol, 1.0 equiv.)], and HATU (66.70 g, 175.39

mmol, 1.05 equiv.). DMSO (506 mL) was then added to the rb flask to give a suspension

[suspension A].

DIPEA (87.28 mL, 501.19 mmol, 3.0 equiv.) was added slowly to suspension A at 23 °C, over

20 minutes to form clear solution. The reaction mixture was stirred at 23 °C for 2.5 h. The

progress of the reaction was monitored by LC/MS. The reaction mixture was added drop wise to

a stirred 3500 mL of cold 1N hydrochloric acid-1N NaCl solution (prepared by adding 204.54 g

of NaCl and 315 mL concentrated hydrochloric acid) to give solid of 3. The solid was dissolved

in EtOAc (1500 mL). The EtOAc layer was washed with water (300 mLx2) followed by brine

(300 mL) and dried over anhyd. Na2SO4. The dried EtOAc layer was filtered and concentrated

under vacuum. The crude product (95.80 g, compound 3, quantitative yield) was analyzed by

LC/MS and used for the next step without further purification.

Synthesis of NH2-Phe-Tyr-(OtBu)-OtBu (4): A 2000 mL rb flask was charged with a stir bar,

(3), 47.5 g, 82.65 mmol], and DCM (275 mL). After dissolving the reaction mixture, Pd/C (10 %

Pd basis, 20% wt/wt, 9.50 g) was added in portions to the rb flask followed by anhyd. MeOH

(551 mL). The reaction mixture was degassed (3x) and H2 gas was bubbled through the reaction

mixture for 4 h under stirring at room temperature. The reaction mixture was filtered through a

Celite plug, washed with MeOH, and the filtrate was concentrated under vacuum to afford 4. The

crude product (36.70 g, compound 4, 99% yield) was analyzed by LC/MS and used for the next

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step without further purification. 1H NMR (500 MHz, Chloroform-d) δ 7.56 (d, J = 7.5 Hz,

1H), 7.29 – 7.14 (m, 5H), 7.00 (dd, J = 8.7, 2.4 Hz, 2H), 6.84 (dd, J = 8.7, 2.3 Hz, 2H), 4.60 (td,

J = 7.5, 5.8 Hz, 1H), 4.06 (t, J = 6.7 Hz, 1H), 3.22 (dd, J = 13.7, 6.2 Hz, 1H), 3.03 (ddd, J = 13.6,

6.3, 3.8 Hz, 2H), 2.92 (dd, J = 13.9, 7.5 Hz, 1H), 1.36 – 1.18 (m, 18H). 13C NMR (125 MHz,

Chloroform-d) δ 170.71, 170.29, 154.07, 135.99, 131.08, 130.03, 129.58, 128.74, 127.14,

124.01, 82.00, 78.25, 55.51, 54.01, 50.39, 39.10, 37.55, 28.79, 27.86.

Synthesis of CbzNH-PEG2-Phe-Tyr(OtBu)-OtBu (6): A 2000 mL rb flask was charged with a

stir bar, Cbz-NH-PEG2-CO2H [(5) 52.90 g, 169.93 mmol, 1.02 equiv.), 4 (73.4 g, 166.598 mmol,

1.0 equiv.), and HATU (66.51 g, 174.93 mmol, 1.05 equiv.). DMSO (505 mL) was then added to

the rb flask under argon and stirred to dissolve. DIPEA (58.04 mL, 333.196 mmol, 2.0 equiv.)

was added slowly to reaction mixture at 23 °C, over 15 minutes. The reaction was stirred at 23

°C for 3 h and progress of the reaction was monitored by LC/MS. The reaction mixture was

added dropwise to a stirred 3000 mL of cold 5% citric acid-1 N NaCl solution (prepared by

adding 175.5 g of NaCl to 3000 mL of 5% Citric acid solution) to ppt as a gummy solid. Filtered

the gummy residue after decanting water and dissolved in EtOAc (1500 mL). The EtOAc layer

was washed with water (300 mL), followed by brine (300 mL), and then dried over anhyd.

Na2SO4. The dried EtOAc layer was filtered and concentrated under vacuum. The crude product

(122.26 g, Compound 6, quantitative yield) was analyzed by LC/MS and used for the next step

without further purification.

Synthesis of NH2-PEG2-Phe-Tyr(OtBu)-OtBu (7): A 2000 mL rb flask was charged with a stir

bar, 6 (61.10 g, 683.29 mmol), and DCM (278 mL). After dissolving the reaction mixture, Pd/C

(10% wt/wt, 12.22 g) was added in portions followed by dry MeOH (556 mL). The reaction

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mixture was degassed (3x) and hydrogen bubbled through reaction mixture for 5 h and then

overnight under hydrogen balloon while stirring at room temperature. Progress of the reaction

was monitored by LC/MS. The reaction mixture was filtered through a Celite plug, washed with

MeOH, and concentrated under vacuum to afford the compound 7. The crude product was used

for the next step without further purification. Compound 7 (50.0 g) was isolated with quantitative

yield.

 Synthesis of DUPA(OtBu)3-NH-PEG2-Phe-Tyr(OtBu)-OtBu (9): A 2000 mL rb flask was

charged with a stir bar, DUPA-(OtBu)3-OH [(8), 77.36 g, 158.34 mmol, 1.0 equiv.], 7 (95.0 g,

158.34 mmol, 1.0 equiv.) and HATU (63.22 g, 166.26 mmol, 1.05 equiv.). DMSO (480 mL) was

then added to the reaction flask under argon. DIPEA (55.2 mL, 316.68 mmol, 2.0 equiv.) was

added slowly to reaction mixture at 23 °C, over 5 minutes. The reaction was stirred at 23 °C for

2.5 h. The product formation was confirmed by LC/MS. The reaction mixture was added drop

wise to a stirred 3000 mL of cold 5% citric acid-1 N NaCl solution (prepared by adding 175.5 g

of NaCl to 3000 mL of 5% Citric acid solution). Filtered the white solid compound and was

dissolved in ethyl acetate (2000 mL) and washed with water (300 mL), followed by brine (300

mL), and dried over anhyd. Na2SO4, filtered and concentrated. Crude product 9 was purified by

silica-gel column chromatography using 60-80% EtOAc in dichloromethane (DCM) followed by

5 % MeOH in DCM. Purified compound 9 was isolated in 88% (148.84 g) yield.

 Synthesis of DUPA-NH-PEG2-Phe-Tyr(OH)-OH (10): A 2000 mL round bottom flask was

charged with a stirring bar and 9 (132 g, 123.32 mmol, 1.0 equiv). A solution of TFA:TIPS:H2O

(95:2.5:2.5, 700 mL) was added to the reaction flask at rt. The reaction mixture was stirred at rt

for 1 h and the progress of the reaction was monitored by LC/MS. The reaction mixture was

evaporated under vacuum (rotavapor) and the concentrated reaction mixture was added drop

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wise to stirred ether (7000 mL) to give white precipitate of 10. The precipitated product was

filtered, washed with cold ether (10 × 1000 mL), and dried under high vacuum to afford 10 as a

white solid. The compound 10 was isolated in 93.4% (91 g) yield. 1H NMR (500 MHz,

Deuterium Oxide) δ 7.22 (t, J = 7.3 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 7.09 (d, J = 7.4 Hz, 2H),

6.97 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.2 Hz, 2H), 4.48 (dd, J = 9.7, 5.2 Hz, 1H), 4.29 (dd, J =

8.2, 4.9 Hz, 1H), 3.94 – 3.86 (m, 2H), 3.54 – 3.35 (m, 6H), 3.33 (t, J = 4.6 Hz, 2H), 3.22 (t, J =

5.5 Hz, 2H), 2.98 (ddd, J = 18.0, 14.1, 5.0 Hz, 2H), 2.79 – 2.66 (m, 2H), 2.37 – 2.24 (m, 2H),

2.24 – 2.08 (m, 4H), 2.00 – 1.85 (m, 2H), 1.75 (tt, J = 12.8, 5.8 Hz, 2H). 13C NMR (125 MHz,

Deuterium Oxide) δ 182.55, 180.21, 179.75, 177.55, 175.88, 173.67, 171.90, 159.09, 154.16,

136.63, 130.64, 129.19, 129.05, 128.65, 127.00, 115.22, 69.27, 68.70, 66.47, 56.23, 55.53, 55.01,

54.66, 38.89, 36.82, 36.78, 35.70, 34.09, 32.41, 29.35, 28.81.

Synthesis of OTL78 (12): A 10 mL rb flask was charged with a stirring bar and 10 (100 g, 127

mmol, 1 equiv). Water (3 mL) was added the rb flask and stirred the suspension (suspension B).

A pH probe is inserted in the suspension B (Initial pH of the suspension = 1.8). A freshly

prepared solution of aq. 1.0 M Na2CO3 was added to suspension B in portions at 23 °C for >5

min until the pH of the reaction solution reaches 10.0 (solution C). Sodium salt of S0456 (11,

115 g, 120.2 mmol, 0.95 equiv.) was added to solution C using solid addition funnel and the

content was stirred till a uniform green suspension is formed (suspension D). Water (1 mL) was

used to wash the chloro-dye from the solid addition funnel in to reaction solution. The flask

containing suspension D was assembled with a condenser, immersed in an oil bath at 70 °C, and

stirred for 3 h. The reaction mixture becomes dark opaque green solution. The reaction was

monitored by LC/MS at every hour until the conversion was ≥98% at 774 nm. The oil bath was

removed and reaction mixture was allowed to cool to ~25 °C while under stirring. The crude

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reaction mixture was diluted with water and purified by RP-HPLC to obtain compound OTL78

(12) with 82% (170.87 g) yield. 1H NMR (500 MHz, Deuterium Oxide) δ 7.70 – 7.60 (m, 4H),

7.57 (d, J = 13.6 Hz, 2H), 7.17 (q, J = 8.7, 7.2 Hz, 5H), 7.06 (d, J = 8.3 Hz, 2H), 6.97 – 6.90 (m,

2H), 6.83 (d, J = 8.1 Hz, 2H), 5.76 (d, J = 13.9 Hz, 2H), 4.64 (s, 1H), 4.34 – 4.19 (m, 2H), 3.90

(dt, J = 8.9, 4.7 Hz, 2H), 3.77 (s, 4H), 3.52 – 3.33 (m, 5H), 3.31 (q, J = 6.9, 5.8 Hz, 1H), 3.24 (dt,

J = 19.6, 4.9 Hz, 4H), 3.13 (d, J = 12.6 Hz, 1H), 2.90 – 2.75 (m, 4H), 2.67 (t, J = 12.5 Hz, 1H),

2.53 (d, J = 12.1 Hz, 1H), 2.41 – 2.08 (m, 11H), 2.00 – 1.83 (m, 3H), 1.74 (tdd, J = 15.2, 11.3,

7.2 Hz, 7H), 1.65 (s, 5H), 1.10 (s, 6H), 1.03 (s, 6H). 13C NMR (125 MHz, Deuterium Oxide) δ

182.57, 180.24, 179.78, 177.72, 175.91, 174.18, 172.26, 172.04, 164.22, 159.11, 158.30, 143.75,

141.83, 141.27, 139.24, 136.41, 132.34, 131.49, 128.75, 127.22, 126.70, 123.62, 119.75, 114.59,

110.67, 100.46, 69.23, 69.16, 68.68, 66.48, 56.25, 55.52, 55.30, 55.03, 50.38, 48.70, 43.50,

38.82, 37.51, 37.15, 35.67, 34.10, 32.40, 29.35, 28.78, 27.28, 26.96, 25.34, 23.72, 21.67, 20.53.

LC/MS (ES+) calcd for C74H93N7O27S4 (free acid) [M+H]+ m/z 1641.8280; found 1641.0980

Biological Studies

In vitro binding of DUPA-FITC: For DUPA-FITC binding affinity, 22Rv1 or PC3 cells were

seeded into a T75 flask and allowed to form a monolayer over 48h. After trypsin digestion, cells

were transferred into centrifuge tubes (1 × 106 cells/tube) and centrifuged. The medium was

replaced with fresh medium containing increasing concentration of DUPA-FITC and incubated

for 30 min at 4 °C. After rinsing with fresh medium (2×1.0 mL) and saline (1×1.0 mL), cells

were re-suspended in saline (1.0 mL) and cell bound fluorescence was analyzed (100,000

cells/sample) using a flow cytometer. The binding affinity (Kd) of DUPA-FITC was calculated

using a plot of percent cell bound fluorescence versus concentration of DUPA-FITC using

GraphPad Prism 6.

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Human Serum Binding Studies: 190 μL of serum was pipetted into 3 separate microcentrifuge

tubes. 190 μL of saline was pipetted into another microcentrifuge tube. 10 μL of OTL78 from 1

mM stock concentration was added to each tube to give a final concentration of 50 μM. 190 μL

of serum was pipetted into 5th microcentrifuge tubes and 10 μL of saline was added to it (blank).

The entire volume of each sample was transferred into a separate Microcon 30 spin filters

(Millipore, MA). Samples were centrifuged at 10, 000 x g for 30 min at room temperature.

Recovered filtrates were analyzed by L/MS.

Safety Study: Seven-week-old healthy male Balb/c (5 mice/ group) were administered with 6

µmol of freshly prepared OTL78 or saline dissolved in 100 µL of saline via tail vein injection on

day zero. Body weights and clinical observations were monitored prior to dosing and daily

thereafter from day zero to 14. Any animals with a body weight loss of 20% or more over two

consecutive days would be euthanized, but this was not necessary.

For immunohistopathology (IHC) studies, the animals were euthanized by CO2 asphyxiation on

the day 14 and selected tissues (brain, heart, lung, liver, spleen, kidney, stomach, small intestine,

large intestine, muscle, and skin) were collected into vials containing 4% formalin. Formalin

fixed tissues were sectioned into 10 μm thick sections and mounted onto Superfrost Plus™ slides

(Fisher Scientific, Pittsburgh PA). After staining the slides with H&E, IHC analysis of the tissues

was conducted to determine to the toxicity of OTL78.

For clinical pathology studies, the animals were euthanized by CO2 asphyxiation on the day 14

and blood was collected to heparin by cardiac punch and blood work analysis was conducted at

Purdue clinical pathology lab.

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Tolerability Studies: The blood samples were collected into hirudin tubes and used within an

hour of collection. For each donor, 4 different tubes were prepared for allergen, positive controls,

and negative control. Samples were analyzed using Flow CAST® high sensitivity Basophil

Activation Test (BAT). Briefly, stimulation buffer (100 μL, background) or anti-FcεR antibody

(100 μL) or fMLP (100 μL) or OTL78 (75 µM in 100 μL of saline) was added to tubes

containing stimulation buffer (200 μL). 100 μL of blood was added to each tube and mixed

gently. After adding staining reagent (40 μL) containing anti-CD63-CD203c-PE-DY647 and

anti-CCR3-PE, each tube was mixed gently and incubated for 15 minutes at 37°C. Lysing

reagent (2 mL) was added to each tube, mixed gently, incubated for 5-10 minutes at room

temperature, and centrifuged for 5 minutes at 500 x g. The supernatant was discarded and the

cells were re-suspended in wash buffer (900 µL) and analyzed using flow cytometry. CD63-

CD203c-PE-DY647+/ CCR3-PE+ cell population considered as the activated basophils.

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