A New Approach for Assessment of Neutrophil Extracellular Traps Through Immunofluorescence Staining in Whole Blood Smears

Materials and reagents

1. APC anti-human CD15 (SSEA-1) (BioLegend, catalog number: 323008)

2. Mouse monoclonal antibody [2C7] to myeloperoxidase (MPO) (Abcam, catalog number: 25989)

3. Rabbit polyclonal antibody to histone H3 (anti-Cit H3 citrulline R2 + R8 + R17) (Abcam, catalog number: 5103)

4. Goat polyclonal antibody to rabbit IgG Alexa Fluor 488 (2 mg/mL) (Abcam, catalog number: ab150077)

5. Goat polyclonal antibody to mouse IgG Alexa Fluor 405 (2 mg/mL) (Abcam, catalog number: ab175660)

6. Sytox Orange nucleic acid stain, 5 mM solution in DMSO (Invitrogen, catalog number: S11368)

7. 10× phosphate buffered saline (PBS), pH 7.2 (HiMedia, catalog number: TL1032-500mL)

8. Tween 20 (Sigma, catalog number: 9005-64-5)

9. Triton X-100 (HiMedia, catalog number: MB031-500mL)

10. Bovine serum albumin (BSA) (HiMedia, catalog number: MB083-100g)

11. Paraformaldehyde (PFA) (Nice Chemicals, catalog number: P64929)

12. Glycerol (≥ 99.5%) (HiMedia, catalog number: MB060)

13. Ultra-pure distilled water

1. Wash buffer (see Recipes)

2. Fixing buffer (see Recipes)

3. Dilution buffer (see Recipes)

4. Permeabilization buffer (see Recipes)

5. Blocking buffer (see Recipes)

6. 70% glycerol (see Recipes)

Recipes

1. Wash buffer (1× PBS)

   Reagent	Final concentration	Quantity or Volume
   10× PBS	1× PBS	5 mL
   Distilled water		~ 45 mL
   HCl	1 M

   Mix 5 mL of 10× PBS with 40 mL of distilled water. Adjust to pH 7.2 with 1 M HCl. Adjust the final volume up to 50 mL with distilled water. Store wash buffer at 4 °C.




2. Fixing buffer

   Reagent	Final concentration	Quantity or Volume
   PFA	4%	10 g
   1× PBS	NA	~250 mL
   NaOH	1 M

   Dissolve 10 g of PFA in 200 mL of 1× PBS in a heating and stirring block (60 °C) and cool down upon complete dissolving of PFA. Add 1 M NaOH solution (4 g of NaOH pellets dissolved in 100 mL of distilled water) dropwise to adjust to pH 6.9. Adjust the final volume up to 250 mL with 1× PBS. Store aliquots at 4 °C (see General note 1).




3. Dilution buffer

   Reagent	Final concentration	Quantity or Volume
   BSA	1%	0.5 g
   1× PBS	NA	50 mL

   Gently mix the solution by inverting the reaction tube. Sterile filter with 0.22 µm pore microfilter. Store at 4 °C.




4. Permeabilization buffer

   Reagent	Final concentration	Quantity or Volume
   1× PBS	NA	100 mL
   Tween 20	0.1%	100 µL
   Triton X-100	0.5%	500 µL

   Prepare this solution in the dark and mix well. Store at 4 °C in air-tight amber bottles, as Tween 20 and Triton X-100 are light-sensitive.




5. Blocking buffer

   Reagent	Final concentration	Quantity or Volume
   BSA	5%	0.5 g
   1× PBS	NA	10 mL

   Gently mix the solution by inverting the reaction tube. Sterile filter with 0.22 µm pore microfilter. Store at 4 °C.




6. 70% glycerol

   Reagent	Final concentration	Quantity or Volume
   Glycerol	70%	35 mL
   1× PBS	NA	15 mL

   Prepare 70% glycerol solution by mixing 35 mL of glycerol with 15 mL of 1× PBS. Use pH paper to ensure a pH of ~7.4. Acidic glycerol will cause rapid fading of fluorochromes. Store at 4 °C in air-tight amber bottles, as glycerol is light sensitive.

Laboratory supplies

1. 15 mL conical centrifuge tubes (Tarsons, catalog number: 546021)

2. 50 mL conical centrifuge tubes (Tarsons, catalog number: 546041)

3. BD Vacutainer K2EDTA vials (BD, catalog number: 454020)

4. BD Emerald^TM single-use syringe, 5 mL (BD, catalog number: 307725)

5. LifeLong^TM 24 G needle, 0.55 mm × 25 mm (Lifelong, catalog number: 021824-H)

6. Microscope slides, 75 mm long × 25 mm wide, thickness 1.35 mm (Blue Star, catalog number: PIC-1)

7. Microscopic cover glass, rectangular (Special) 22 mm × 60 mm (Blue Star, catalog number: 5128789)

8. Pierce^TM microcentrifuge tubes, 1.5 mL (Thermo Scientific, catalog number: 69715)

9. Pierce^TM microcentrifuge tubes, 2.0 mL (Thermo Scientific, catalog number: 69720)

10. Syringe-driven filters, filter pore size 0.22 µm, diameter 30 mm (HiMedia, catalog number: SF137-100NO)

11. Advanced PAP Pen, 5 mm tip width (Sigma, catalog number: Z377821)

12. Borosilicate glass graduated round reagent bottles with screw caps (Borosil, catalog number: 1519)

13. Graduated cylinder (Borosil, catalog number: 3021)

14. Humidifier slide chamber (we used customized humidifier slide chamber of dimensions 35 cm × 30 cm × 6 cm from Seven Star Scientific Instruments. Researchers can also use Evergreen Scientific slide moisture chamber of dimensions 8^1/4×7×1^1/4" from VWR, catalog number: 76278-832) (see General note 4)

15. Qualigens Labolene Neutral pH 5 L (Thermo Fisher Scientific, catalog number: Q42218)

16. Sterile pipette tips 0.5–10 µL, 1–200 µL, 100–1,000 µL volume range (Tarsons)

Equipment

1. Micro-pipettes 0.5–10 µL, 10–100 µL, 20–200 µL, 100–1,000 µL (Eppendorf)

2. Class II biological safety cabinet (ESCO Lifesciences, model: AC2-4S8-NS)

3. SPINOT^TM Digital Magnetic Stirrer Hot Plate (Tarsons, model: MC 02, Catalogue number: 6040)

4. Olympus Fluoview confocal laser scanning microscope (Olympus, model: FV3000)

5. RO-DI Ultra (Rions Labpure water solutions, model: ASTM Type I)

Software and datasets

1. ImageJ; Java-based program (1.5.4) (https://imagej.nih.gov/ij/download.html)

2. Microsoft Office Professional Plus 2019

Procedure

1. Collect pre-operative blood samples of PDAC patients and healthy controls in EDTA vials (all the patients/participants provided written, informed consent for their participation in this study).

2. With the help of a micro-pipette, put a sample of 2 μL of whole blood on a glass slide cleaned with 90% ethanol. Place the blood sample 1 cm away from one edge of the glass slide as illustrated in Figure 1.

   
   

   
   Figure 1. Stepwise protocol for whole-blood smear preparation. a. Clean the slides with 90% alcohol. b. Place 2 μL EDTA-treated blood on a slide with pipette. c. Position the spreader at a 45° angle on the blood drop and through a capillary mechanism, blood will spread along the edge in contact with the spreader. Move the spreader forward quickly, straight, and smoothly along the length of the slide. d. Make the boundary with advanced PAP pen around the smear to prevent the buffers spill out from the slide. e. Permit the smear to air-dry for 15 min. f. Position the slide above the racks of humidifier slide chamber.

   
   

3. Position the spreader at a 45° angle in front of the blood drop. Push it back until it makes contact with the blood. Through a capillary mechanism, blood will spread along the edge in contact with the spreader. Move the spreader forward quickly, straight, and smoothly along the length of the slide (see General note 2).

4. Create a perimeter using the hydrophobic advanced PAP pen around the smear to prevent the spilling out of buffers from the slide. Allow the smear to air-dry naturally for 15 min at room temperature. Be cautious of excessive air-drying, as it can damage proteins and cellular structures, potentially affecting immunodetection outcomes.

5. Apply 200 μL of fixing buffer onto the smear using a micro-pipette to fix the samples and place them in a humidifier slide chamber for 10 min (see General note 3).

6. Tilt the slide gently to allow the excess PFA liquid to drain into the waste container. Following this, wash the slide three times with wash buffer (1 mL each time) and dispose of these washes in the same waste container to ensure proper disposal of toxic PFA residues.

7. Apply 200 μL of APC-labeled CD15 antibody diluted with dilution buffer and incubate in the dark for 30 min at room temperature in a humidifier slide chamber. Remove excess antibody by rinsing the slides three times with 1 mL of wash buffer and gently tilt the slides to discard the wash liquid into the waste container (see General note 4).

8. Apply 200 μL of permeabilization buffer to permeabilize the slides for 30 min at room temperature in the dark and wash the slides with 1 mL of wash buffer three times.

9. Apply 200 μL of blocking buffer on the smear for 30 min at room temperature in the dark to block the sections and then rinse the slides three times with 1 mL of wash buffer.

10. Apply 200 μL of diluted (as listed in Table 1) anti-MPO and anti-Cit H3 primary antibodies using the dilution buffer. Let the slides incubate in the dark for 3 h at room temperature in a humidifier slide chamber to avoid the tissue drying out and then wash the slides three times with 1 mL of wash buffer.

    
    

    Table 1. Stock and working volume of antibodies

    Antibody	Stock solution	Working solution
    APC-labeled CD15	1 mg/mL	1:200
    Cit H3	1 mg/mL	1:500
    MPO	1 mg/mL	1:500
    Goat anti-rabbit Alexa Fluor 488	2 mg/mL	1:1,000
    Goat anti-mouse Alexa Fluor 405	2 mg/mL	1:1,000
    Sytox Orange	5 mM	1:1,500




11. Apply 200 μL of goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 405 secondary antibodies diluted 1:1,000 in dilution buffer. Let the slides incubate in the dark for 1 h at room temperature in a humidifier slide chamber and wash the slides three times with 1 mL of wash buffer.

12. Stain slides with 5 mg/mL of Sytox Orange diluted 1:1500 in wash buffer for 5 min in the dark at room temperature. After staining, wash three times using 1 mL of wash buffer.

13. Apply a drop (20–30 μL) of 70% glycerol to the edge of the smear in the dark and then gently lower the coverslip onto the slide from that side, making sure there are no bubbles (see General note 5).

14. Let the slides dry for at least 15 min in a dark environment. Then, proceed with imaging of the slides using the Olympus Fluoview FV3000 confocal microscope.

Controls

Two types of negative controls can be utilized for quantifying positive staining of NETs:

1. Isotype control antibodies (mouse IgG and rabbit IgG) as a negative control in place of anti-MPO, and anti-citrullinated Histone 3 for primary staining at similar doses. As an alternative, incubate the slides in the host-specific serum in place of the primary antibodies (such as mouse and rabbit sera against MPO and citrullinated Histone 3 antibodies). The secondary staining is performed as described above.

2. Stain secondary antibodies in a manner akin to this, but without the addition of primary antibodies.

Data analysis

1. Image acquisition

   Acquire images with a confocal microscope (Olympus Fluoview FV3000 in this case). Choose the suitable lasers; in this instance, 405 nm (MPO), 488 nm (Cit H3), 650 nm (CD15), and 555 nm (Sytox Orange). For identification of stained structures, adjust the voltages of the laser. Make sure staining is specific by comparing positive samples to control samples. After the lasers have been adjusted, utilize the same parameters for each slide you need to compare and examine. Extracellular structures that stain positively for CD15, MPO, and Cit H3 are referred to as NETs, illustrated in Figures 2 and 3.

   
   

   
   Figure 2. Representative images of neutrophil extracellular traps in peripheral blood smears of healthy controls. Individual images represent the positive staining for nuclear binding dye Sytox Orange (555 nm, red), CD15 (650 nm, magenta), MPO (405 nm, blue), Cit H3 (488 nm, green), and other combinations of CD15, Cit H3, and MPO, along with nuclear dye. Neutrophil extracellular traps are identified as extracellular structures where nuclear binding dye Sytox Orange (555 nm, red), colocalized with CD15 (650 nm, magenta), MPO (405 nm, blue), and Cit H3 (488 nm, green) in the merged image as indicated by the white arrows. Images acquired at 40× magnification, scale bars = 60 µm.

   
   

   
   Figure 3. Representative images of neutrophil extracellular traps in peripheral blood smears of pancreatic cancer patients. Individual images represent the positive staining for nuclear binding dye Sytox Orange (555 nm, red), CD15 (650 nm, magenta), MPO (405 nm, blue), Cit H3 (488 nm, green), and other combinations of CD15, Cit H3, and MPO, along with nuclear dye. Neutrophil extracellular traps are identified as extracellular structures where nuclear binding dye Sytox Orange (555 nm, red), colocalized with CD15 (650 nm, magenta), MPO (405 nm, blue), and Cit H3 (488 nm, green) in the merged image as indicated by the white arrows. Images acquired at 40× magnification, scale bars = 60 µm.




2. Quantitation of NETs

   To further quantify the NETs, analyze the slides through confocal microscopy. Visualize the slides and take images of each slide at three high-power random fields, i.e., 40× magnification. Open the images in ImageJ and split the image into channels. Visualize the mentioned channels, i.e., blue (MPO), green (Cit H3), magenta (CD15), and red (Sytox Orange). The overlaid channels, which are positive for all markers (MPO, Cit H3, CD15, and Sytox Orange) represent the NETs. Count the NET-positive cells (CD15+, MPO+, and Cit H3+) and their subtypes (CD15+ MPO+ and CD15+ Cit H3+) in the peripheral blood smear images of both control and PC patients. Calculate the relative percentage of NETs and their subtypes as shown in Figure 4.

   Note: Alternatively, researchers can use different immunofluorescence-based reporting methods available in literature such as fluorescence intensity to report the presence of markers.

   
   

   
   Figure 4. Comparison of neutrophil extracellular trap markers in peripheral blood smears. The bar graph indicates the relative percentage of neutrophils (CD15+), neutrophil extracellular traps (CD15+, MPO+, and Cit H3+), and their subtypes (CD15+ MPO+ and CD15+ Cit H3+) in relation with total Sytox positive cells in the peripheral blood smears of both control and pancreatic cancer patients. The x-axis represents the markers of neutrophil extracellular traps in the peripheral blood smears of both control and pancreatic cancer patients, whereas y-axis represents the relative percentage of various markers of neutrophil extracellular traps in relation with total Sytox positive cells. Data in bar graphs is represented in the form of mean ± standard error of mean, n = 5. PC, pancreatic cancer.

Validation of protocol

This protocol is a part of our ongoing research work on the role of neutrophil extracellular traps in promoting epithelial–mesenchymal transition and immunomodulation in pancreatic cancer. We have followed a standardized methodology to identify and characterize NETs in whole blood smears and provided every minute details regarding the same. The protocol will be cited accordingly in future publications. For related protocols, researchers can also refer to other immunofluorescence-based protocols for NETs detection performed in plasma samples (Matta, B., Battaglia, J. and Barnes, B. J., 2022. Detection of neutrophil extracellular traps in patient plasma: method development and validation in systemic lupus erythematosus and healthy donors that carry IRF5 genetic risk. Frontiers in immunology, 13, 951254. https://doi.org/10.3389/fimmu.2022.951254 [18]).

General notes and troubleshooting

General notes

1. Safety precautions must be taken when handling PFA, which is toxic and carcinogenic, and for NaOH pellets, which are highly corrosive. Prepare these solutions in a well-ventilated environment, wear a lab coat, gloves, safety glasses, and other protective gear, and avoid direct contact with these chemicals.

2. A spreader should be thinner than a glass slide when it comes to spreading blood on slides. The spreader's edge ought to be polished, smooth, and slim. The smear ought to extend halfway over the slide. It must be devoid of gaps, cracks, and bubbles. The smear should be smooth, leveled, and slightly curved in appearance. It must be sufficiently thin to produce a low power field of at least 10× in areas without the RBCs overlapping. Avoid applying too much pressure when spreading, and let the smear air-dry fully before staining in order to prevent RBC overlap. During staining, drying aids in preventing RBC overlapping.

3. To maintain high humidity, add 10 mL of water to the humidifier slide chamber. Slides should be placed atop the racks of the humidifier slide chamber. To preserve the moisture inside the container, close the lid of the humidifier slide chamber.

4. For washing, rinse the slides three times with 1 mL of wash buffer (each time gently without shaking) and gently tilt the slides to remove the liquid into the waste container.

5. Commercially available anti-fade mounting media can also be used instead of 70% glycerol. A refractive index (RI) of approximately 1.53 is suggested for the mounting medium. The brightness and clarity of the image increase with the proximity of the sample's and mounting medium's RI values to this value. With an RI of 1.47, glycerol boosts the sample's RI and helps in the emergence of a brighter, better-resolution image.

Troubleshooting

Acknowledgments

We appreciate the engagement of every patient and volunteer. A Ph.D. scholarship from the Department of Biotechnology, Government of India, to Sakshi Bansal, helped with this work during the protocol's optimization (Award letter Number DBTHRDPMU/JRF/BET-21/I/2011-22/251). We express our gratitude to Ms. Meenakshi Sinha for her help with confocal microscopy, the Central Sophisticated Instrumentation Core (CSIC), and PGIMER, Chandigarh for their infrastructure support.

Competing interests

There are no competing interests to declare.

Ethical considerations

All described procedures for sample collection were consented to by the Institutional Ethics Committee of the Post Graduate Institute of Medical Education and Research, Chandigarh on February 27, 2023 (reference number IEC-INT/2023/PhD-885). All the patients/participants provided written, informed consent for their participation in this study.

References

1. Yabroff, K. R., Wu, X. C., Negoita, S., Stevens, J., Coyle, L., Zhao, J., Mumphrey, B. J., Jemal, A. and Ward, K. C. (2021). Association of the COVID-19 Pandemic With Patterns of Statewide Cancer Services. J Natl Cancer Inst. 114(6): 907–909.
2. Siegel, R. L., Miller, K. D., Fuchs, H. E. and Jemal, A. (2022). Cancer statistics, 2022. CA Cancer J Clin. 72(1): 7–33.
3. Hu, J. X., Zhao, C. F., Chen, W. B., Liu, Q. C., Li, Q. W., Lin, Y. Y. and Gao, F. (2021). Pancreatic cancer: A review of epidemiology, trend, and risk factors. World J Gastroenterol. 27(27): 4298–4321.
4. Gordon‐Weeks, A. N., Lim, S. Y., Yuzhalin, A. E., Jones, K., Markelc, B., Kim, K. J., Buzzelli, J. N., Fokas, E., Cao, Y., Smart, S., et al. (2017). Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2–dependent angiogenesis in mice. Hepatology. 65(6): 1920–1935.
5. Teramukai, S., Kitano, T., Kishida, Y., Kawahara, M., Kubota, K., Komuta, K., Minato, K., Mio, T., Fujita, Y., Yonei, T., et al. (2009). Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: An analysis of Japan Multinational Trial Organisation LC00-03. Eur J Cancer. 45(11): 1950–1958.
6. Schmidt, H., Suciu, S., Punt, C. J., Gore, M., Kruit, W., Patel, P., Lienard, D., von der Maase, H., Eggermont, A. M., Keilholz, U., et al. (2007). Pretreatment Levels of Peripheral Neutrophils and Leukocytes As Independent Predictors of Overall Survival in Patients With American Joint Committee on Cancer Stage IV Melanoma: Results of the EORTC 18951 Biochemotherapy Trial. J Clin Oncol. 25(12): 1562–1569.
7. Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D. S., Weinrauch, Y. and Zychlinsky, A. (2004). Neutrophil Extracellular Traps Kill Bacteria. Science. 303(5663): 1532–1535.
8. Mohanan, S., Cherrington, B. D., Horibata, S., McElwee, J. L., Thompson, P. R. and Coonrod, S. A. (2012). Potential Role of Peptidylarginine Deiminase Enzymes and Protein Citrullination in Cancer Pathogenesis. Biochem Res Int. 2012: 1–11.
9. Jin, W., Yin, H., Li, H., Yu, X., Xu, H. and Liu, L. (2021). Neutrophil extracellular DNA traps promote pancreatic cancer cells migration and invasion by activating EGFR/ERK pathway. J Cell Mol Med. 25(12): 5443–5456.
10. Demers, M., Wong, S. L., Martinod, K., Gallant, M., Cabral, J. E., Wang, Y. and Wagner, D. D. (2016). Priming of neutrophils toward NETosis promotes tumor growth. Oncoimmunology. 5(5): e1134073.
11. Kanamaru, R., Ohzawa, H., Miyato, H., Matsumoto, S., Haruta, H., Kurashina, K., Saito, S., Hosoya, Y., Yamaguchi, H., Yamashita, H., et al. (2018). Low density neutrophils (LDN) in postoperative abdominal cavity assist the peritoneal recurrence through the production of neutrophil extracellular traps (NETs). Sci Rep. 8(1): 632.
12. Demkow, U. (2021). Neutrophil Extracellular Traps (NETs) in Cancer Invasion, Evasion and Metastasis. Cancers (Basel). 13(17): 4495.
13. Cools-Lartigue, J., Spicer, J., McDonald, B., Gowing, S., Chow, S., Giannias, B., Bourdeau, F., Kubes, P. and Ferri, L. (2013). Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest. 123(8): 3446–3458.
14. Martins-Cardoso, K., Almeida, V. H., Bagri, K. M., Rossi, M. I. D., Mermelstein, C. S., König, S. and Monteiro, R. Q. (2020). Neutrophil Extracellular Traps (NETs) Promote Pro-Metastatic Phenotype in Human Breast Cancer Cells through Epithelial–Mesenchymal Transition. Cancers (Basel). 12(6): 1542.
15. Albrengues, J., Shields, M. A., Ng, D., Park, C. G., Ambrico, A., Poindexter, M. E., Upadhyay, P., Uyeminami, D. L., Pommier, A., Küttner, V., et al. (2018). Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science. 361(6409): eaao4227.
16. Langiu, M., Palacios-Acedo, A. L., Crescence, L., Mege, D., Dubois, C. and Panicot-Dubois, L. (2022). Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research. Int J Mol Sci. 23(3): 1257.
17. Boone, B. A., Murthy, P., Miller-Ocuin, J., Doerfler, W. R., Ellis, J. T., Liang, X., Ross, M. A., Wallace, C. T., Sperry, J. L., Lotze, M. T., et al. (2018). Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. BMC Cancer. 18(1): 678.
18. Matta, B., Battaglia, J., Barnes, B. J. (2022). Detection of neutrophil extracellular traps in patient plasma: method development and validation in systemic lupus erythematosus and healthy donors that carry IRF5 genetic risk. Front Immunol. 13: 951254.