A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma

Materials and reagents

1. Poly-L-Lysine adhesive microscope slides (Newcomer Supply, catalog number: 5010)

2. Immulon^TM Microtiter^TM 96-well plates (Thermo Scientific, catalog number: 3855)

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

4. Rabbit polyclonal antibody to histone H3 (anti-CitH3 citrulline R2 + R8 + R17) (Abcam, catalog number: 5103)

5. Sytox Green nucleic acid stain (Invitrogen, catalog number: S7020)

6. Cell Death Detection ELISA PLUS Anti-DNA POD kit (Roche, catalog number: 11774425001)

7. Goat anti-mouse Alexa Fluor 488 (2 mg/mL) (Invitrogen, catalog number: A11029)

8. Goat anti-rabbit Alexa Fluor 594 (2 mg/mL) (Molecular Probes, catalog number: A11012)

9. DAPI (4’,6-Diamidino-2-Phenylindole, dihydrochloride) (Sigma-Aldrich, catalog number: D9542-1MG)

10. TMB (3,3’,5,5’-tetramethylbenzidine) enhanced one component HRP membrane substrate (Sigma-Aldrich, catalog number: T9455)

11. 2N sulfuric acid stop solution (Reagents, catalog number: CS106300-500A)

12. Normal rat serum control (Fisher Scientific, catalog number: 10-710-C)

13. Tween 20 (Thermo Fisher, catalog number: J20605.AP)

14. Triton X-100 (Thermo Fisher, catalog number: HFH10)

15. Bovine serum albumin (BSA) (Fisher catalog number: BP9703-100)

16. Formaldehyde (Sigma, catalog number: 252549)

17. Na₂CO₃ (Sigma, catalog number: 497-19-8)

18. NaHCO₃ (Sigma, catalog number: 144-55-8)

19. VectaMount AQ mounting medium (Vector Labs, catalog number: H-5501)

20. Dulbecco’s Phosphate Buffer saline (PBS without calcium and magnesium chloride (Sigma, catalog number: D8537-500ML)

21. Wash buffer (see Recipes)

22. Blocking buffer (see Recipes)

23. Dilution buffer (see Recipes)

24. Coating buffer (see Recipes)

25. Smear dilution buffer (see Recipes)

26. Smear blocking buffer (see Recipes)

27. Fixing buffer (see Recipes)

Equipment

1. Pipettes, multi-channel pipettes for washings and tips

2. Synergy Neo2 Multi-Mode microplate reader (BioTek, model: BTNEO2)

3. EVOS M7000 imaging system (Invitrogen, catalog number: AMF7000)

4. ZEISS Confocal LSM 880 Airyscan

5. NanoDrop 2000 (Thermo Fisher Scientific, catalog number: ND-2000)

Software

1. ImageJ—Java-based program (V1.8) (https://imagej.nih.gov/ij/download.html)

2. GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/)

3. MyAssays online program (https://www.myassays.com/download-and-install-myassays-desktop.html)

Procedure

1. NETs ELISA

   1. Coat 96-well ultra-high-binding plates with 50 μL of anti-MPO and anti-CitH3 unlabeled primary antibodies diluted to a concentration of 5 μg/mL each in coating buffer. Cover the plate with the plate sealer. Leave plate overnight at 4 °C.

   2. The next morning, wash wells three times with 200 μL of PBS at room temperature.

   3. Block plate with 200 μL of blocking buffer for 2 h at room temperature.

   4. Wash the plate five times with 200 μL of PBS.

   5. Add 50 μL of undiluted plasma sample to each coated well and incubate at room temperature for 2 h. Set up wells for blanks where no plasma samples are added.

   6. Carefully wash the plate five times with 200 μL of wash buffer; do not invert plate during washes.

   7. Add 50 μL of anti-DNA-POD antibody (from Cell Death Detection ELISA PLUS Anti-DNA POD kit) prepared 1:100 in dilution buffer to each well and incubate at room temperature for 2 h for the detection of NETs.

   8. Wash the plate five times with 200 μL of wash buffer.

   9. Add 100 μL of TMB (allow to come to room temperature before using) to each well and incubate plate in the dark for 3 min.

   10. Add 100 μL of 2 N sulfuric acid stop solution to each well and measure absorbance using a Synergy Neo2 Multi-Mode microplate reader, or any ELISA microplate reader set to a wavelength of 450 nm.




2. Smear assay

   1. Draw a circle using a histology liquid repellent pen on a poly-L-Lysine glass slide (2–3 circles per slide).

   2. Drop 1 μL of plasma sample to each circle and smear in a circular motion until the volume is equally distributed. Let plasma completely dry.

   3. Fix samples with 100 μL of smear fixing buffer (4% formaldehyde) for 10 min.

   4. Carefully tilt the slide to remove the liquid to be collected in the formaldehyde waste container, dab the sides of the slides with a paper towel, and carefully wash each circle three times with 100 μL of PBS.

   5. Block the slides with 100 μL of smear blocking buffer (5% BSA/PBS) for 1 h at room temperature.

   6. Add 50–100 μL of anti-MPO and anti-CitH3 primary antibodies diluted to smear dilution buffer (1 μg/mL in 0.3% Triton X-100 + 0.1% BSA solution) to each circle and leave overnight in a moist slide chamber.

   7. The following morning, wash slides three times with PBS and add 50–100 μL of goat anti-mouse Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 secondary antibodies diluted 1:500 in dilution buffer to each circle and incubate in the dark for 1 h.

   8. Wash three times with 200 μL of PBS and stain slides with 5 mg/mL of DAPI diluted 1:200 in PBS for 15 min in the dark.

   9. In another set of slides for quick nonspecific visualization, after fixing and blocking, NETs can be stained directly with nuclear dyes Sytox Green diluted 1:200 and 5 mg/mL of DAPI diluted 1:200 in PBS for 15 min in the dark.

   10. Wash an additional three times with 200 μL of wash buffer after staining. Add a drop of VectaMount AQ mounting medium and secure a glass coverslip onto the slide making sure that there are no bubbles.

   11. Allow the slides to dry before imaging on EVOS M7000 imaging system and/or ZEISS Confocal LSM 880.

   12. Stain secondary antibody–only controls the similar way, except that no primary antibodies are added.

Data analysis

For ELISA data analysis, a standard curve is prepared using serial dilutions of three pooled phorbol 12-myristate 13-acetate (PMA)-stimulated healthy donor plasma samples. DNA is quantified using the NanoDrop 2000. NETs in serially diluted PMA-induced plasma samples were quantified using CitH3 and MPO + CitH3 ELISA methodology. Four-parameter logistic standard curves were created for each antibody coating, and data were extrapolated using MyAssays online software. A detailed protocol can be found in the published manuscript (Matta et al., 2022) in methodology under section “Preparing in-house standard for the quantification of NET remnants by ELISA.”

For quantification of NETs in smear assay we used ImageJ Java-based program (V1.8) to convert the images to grayscale 8-bit images, whose intensities were then quantified and graphed. A detailed protocol can be found in the published manuscript (Matta et al., 2022) in methodology under section “NET Quantification using ImageJ.”

All statistical analyses should be performed and graphed using GraphPad Prism 8. A two-tailed parametric t-test should be used for comparisons between samples with normal distribution. P < 0.05 should be considered statistically significant. A two-tailed correlation analysis should be performed using Pearson correlation coefficients assuming Gaussian distribution.

Notes

There is a disparity between ELISA and smear assay when using fresh vs. frozen plasma samples. ELISA can detect NETs in circulation from samples stored at -80 °C for long periods of time (> 6 months), as it can detect both intact and fragmented NETs. But as the smear assay is visualizing intact NETs, we found that NET structures are generally maintained in plasma samples up to six months when stored at -80 °C. Beyond six months, we could detect fragmented NETs with smear assay that compromised their quantification. According to our preliminary studies, we suggest adding EDTA/EGTA immediately after plasma isolation to prolong the stability of intact NET structures for quantification. Although we used the anti-DNA-POD antibody from Cell Death Detection ELISA PLUS Anti-DNA POD kit for the detection of NETs in ELISA, we believe that any other anti-DNA-peroxidase-conjugated antibody can be used for detection.

Recipes

1. Wash buffer

   1% BSA + 0.05% Tween 20 in PBS




2. Blocking buffer

   5% BSA + 5% normal rat serum in PBS




3. Dilution buffer

   5% BSA + 0.05% Tween 20 in PBS




4. Coating buffer

   15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6

   To 50 mL of deionized water, add 80 mg of Na₂CO₃ and 150 mg of NaHCO₃




5. Smear dilution buffer

   0.3% Triton X-100 + 0.1% BSA in PBS




6. Smear blocking buffer

   5% BSA in PBS




7. Fixing buffer

   4% formaldehyde in PBS

Acknowledgments

This work was supported by grants from the National Institutes of Health NIAMS 1 R01 AR 076242-03, Department of Defense CDMRP LRP W81XWH-18-1-0674, and Lupus Research Alliance to BJB.

We would also like to thank members of “The NETwork to Target Neutrophils in COVID-19” for inspiration to develop new methodologies to detect NETosis.

This protocol was derived from the original work of Matta et al. (2022).

Graphical overview made with the help of https://biorender.com/.

Competing interests

The authors declare no conflicts of interest.

Ethics

The studies involving human participants were reviewed and approved by The Feinstein Institutes for Medical Research IRB. The patients/participants provided their written informed consent to participate in this study.

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