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