Reagents

NETosis by Flow Cytometry Protocol

Media:

D10 media = DMEM, 10% FBS

FACS media = PBS, 2% FBS, 0.02% NaAz

Reagents:

Sytox Blue = Helix NPTM Blue, Biolegend, #425305

Live/Dead UV = Live/DeadTM Fixable Blue Dead Stain Kit, Fisher, #L23105 Fc Block = Purified Anti-Mouse CD16/CD32 (2.4G2), Tonbo, #70-0161-M001 αCD11b = PE-Cyanine7 anti-Human/Mouse CD11b, Tonbo, #60-0112-U100 αLy6G = Brilliant Violet 711TM anti-mouse Ly-6G, BioLegend, #127643

αmyeloperoxidase = Anti-myeloperoxidase antibody [2D4] (Biotin), Abcam, #ab90811 αcitrullinated Histone = Anti-histone H3 (citrulline R2+R8+R17) rabbit antibody, Abcam, #ab5103 Streptavidin A488 = Alexa Fluor® 488 Streptavidin, BioLegend, #405235

αRabbit A647 = Alexa Fluor® 647 Donkey anti-rabbit IgG Antibody, BioLegend, #406414

Protocol:

1. Plate 2.5x105 neutrophils into 96-well low round bottom low cluster (Costar, #7007). Bring the volume up to 200 µL using D10. Let cells rest in the incubator for 2 hr.
2. Add 100 μL of an overnight S. aureus culture to 900 μL non-heat inactivated serum. Place on ice for 2 hr.
3. Add bacteria (MOI = 10) to neutrophils for 30, 60, 120 or 240 min (synchronize so time points end simultaneously).
4. 20 min prior to the end of the time points, add Sytox Blue and Live/Dead stain to the samples.
   1. Sytox Blue (1:1000)
   2. Live/Dead UV (1:1000)
5. Once time points end, spin, aspirate, and fix cells by adding 100 μL room temperature 4% PFA (in PBS) for 15 min on ice.
6. Spin, aspirate, and add 100 μL cold Fc Block (1:150 in FACS media) for 20 min on ice.
7. Spin, aspirate, and add 100 μL cold primary stain in FACS media for 20 min on ice. 
   i. αCD11b PE/Cy7 (1:800)

ii. αLy6G (1:800)

iii. αmyeloperoxidase Biotin (1:200)

iv. αcitrullinated histone H3 rb (1:200)

    8. Spin, aspirate, add 100 μL cold 2° stain in FACS media for 20 min on ice.

1. Streptavidin A488 (1:1000)

2. αRabbit A647 (1:1000)

    9. Spin, aspirate, and transfer samples to flow tubes (1.2 mL Micro Titer Tubes, Fisher, #02-681-376) in cold FACS media. Keep in the fridge until running samples.

   10. Gate samples side scatter height (SSC-H) by side scatter area (SSC-A) followed by forward scatter height (FSC-H) by forward scatter area (FSC-A) to remove doublet populations. The singlet population is gated SSC-A by FSC-A to isolate the granulocyte population. From there the following gating is performed to identify neutrophils undergoing vital or suicidal NET formation:

11. Calculate the percentage of neutrophils (Ly6G⁺CD11b⁺) undergoing vital (live: extracellular dsDNA⁺MPO⁺H3Cit⁺) or suicidal (dead: extracellular dsDNA⁺MPO⁺H3Cit⁺) NET formation by dividing the number cells in gate 2 or 3 by the total number of neutrophils (gate 1).

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