Neutrophils were plated at 5 × 105 cells per well in complete RPMI medum in a 96-well tissue culture plate. Where appropriate, wells were treated with PRT at a concentration of 2 μM. Candida species at an MOI of 10 were coincubated with the neutrophils for 1 h at 37°C. After the incubation, Sytox red (Invitrogen, Carlsbad, CA) was added at a final concentration of 160 nM to detect extracellular DNA. Sytox red fluorescence was read every hour for 18 h at 30°C using a SpectraMax i3x reader, and the results were expressed as arbitrary fluorescence units. Staining for NET histones was carried out as previously described (69 (link)). Briefly, neutrophils were incubated with C. albicans hyphae or C. glabrata and C. auris yeast cells for 3 h. Samples were then stained with anti-histone H3 citrulline R2+R8+R17 (Abcam, Cambridge, MA; 0.014 mg/ml) followed by donkey anti-rabbit IgG Cy3 (Jackson Immunoresearch, West Grove, PA; 0.0075 mg/ml) secondary antibody. Samples were then imaged at ×40 on Nikon Ti-E microscope.
Anti histone h3 citrulline r2 r8 r17
Manufactured by Abcam
Sourced in United States
Anti-Histone H3 (citrulline R2 + R8 + R17) is a lab equipment product that specifically binds to histone H3 proteins with citrullinated arginine residues at positions 2, 8, and 17. This antibody can be used to detect and quantify these modified histone proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop 2 plus, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (2 mentions)
Anti p47, by Santa Cruz Biotechnology (2 mentions)
Anti neutrophil elastase, by Abcam (2 mentions)
Anti human mouse myeloperoxidase mpo, by R&D Systems (2 mentions)
Anti c albicans, by Merck Group (2 mentions)
Anti p40, by Merck Group (2 mentions)
Anti cd63, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (2 mentions)
Superfrost plus micro slide, by Avantor (2 mentions)
Cy3 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Sytox red, by Thermo Fisher Scientific (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Anti γh2ax antibody, by Cell Signaling Technology (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Paraffin, by Leica (1 mentions)
Fluoview 3000, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
9 protocols using anti histone h3 citrulline r2 r8 r17
1
Neutrophil Antifungal Defense Mechanisms
2
Detecting DNA Damage and Histone Modifications
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Each group of cells (5 × 104) was incubated with 2.22 MBq or 18.5 MBq of I-131 for 7 h and then fixed in methanol at −20 °C for 5 min. After blocking with normal goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min, cells were incubated with the following primary antibodies at 4 °C overnight: anti-γ-H2AX antibody (Cell Signaling, Danvers, MA, USA) and anti-histone H3 (citrulline R2 + R8 + R17) (Abcam, Cambridge UK; ab5103). The cells were washed and stained with Alexa Fluor 555 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature and then mounted with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA). Fluorescence was observed using a confocal microscope (Carl Zeiss, Oberkochen, Germany).
3
Histological and Immunofluorescent Analysis of Cardiac Tissue
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Hearts were embedded in optimal cutting temperature compound (O.C.T.; Sakura® Finetek Japan Co., Ltd., Tokyo, Japan) or in paraffin (Leica Biosystems, Wentzler, Germany). 5 μm thick paraffin sections were used for stained with haematoxylin and eosin (H&E) and Masson's trichrome staining according to standard procedures.
Immunofluorescent staining was performed to detect neutrophil infiltration (neutrophil elastase and α-actinin, Abcam, Cambridge, UK) and NETs formation [anti-histone H3 citrulline R2+R8+R17, and myeloperoxidase (MPO), Abcam]. Co-staining was visualized by fluorescence microscope with 488- and 596-conjugated secondary antibodies (Abcam) mounted with fluoroshield mounting medium with 4ʹ,6-diamidino-2-phenylindole (DAPI, Abcam).
Immunofluorescent staining was performed to detect neutrophil infiltration (neutrophil elastase and α-actinin, Abcam, Cambridge, UK) and NETs formation [anti-histone H3 citrulline R2+R8+R17, and myeloperoxidase (MPO), Abcam]. Co-staining was visualized by fluorescence microscope with 488- and 596-conjugated secondary antibodies (Abcam) mounted with fluoroshield mounting medium with 4ʹ,6-diamidino-2-phenylindole (DAPI, Abcam).
4
Neutrophil Immunofluorescence Staining
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5×104 human peripheral neutrophils were plated on glass coverslips 1 h prior to stimulation. After stimulation, cells were fixed with 2% paraformaldehyde for 20 min at 37°C. Immunofluorescence staining was performed as described elsewhere. Anti-neutrophil elastase (NE) (Abcam), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Life technologies), anti-human/mouse myeloperoxidase (MPO) (R&D Systems), anti-C. albicans (Acris), anti-p40 (Millipore, 1.22), anti-p47 (Santa Cruz), anti-CD63 (Millipore, RFAC4). For lung histology dissected lungs of infected mice were fixed over night in 2% paraformaldehyde and embedded in wax for sectioning. Sections were treated with a standard antigen retrieval protocol and immunofluorescence staining as described elsewhere. Cit-H3: anti-Histone H3 (citrulline R2 + R8 + R17) (Abcam, ab5103), MPO, DAPI. Stained cells and tissues were mounted and examined with confocal microscopy. Image analysis was performed using ImageJ.
5
Neutrophil Immunofluorescence Visualization
6
Histone Citrullination in Sperm Chromatin
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After sperm prep, samples were dried onto VWR Superfrost Plus Micro Slides (48311–703), fixed for 10 minutes in 4% paraformaldehyde solution, washed 3x with PBS, and permeabilized for 10 min with 0.1% triton in PBS. Blocking was performed with 10% BSA in PBS for 1 hour at room temperature in a humidified chamber. Staining with primary antibody, Anti-Histone H3 (citrulline R2 + R8 + R17 (Abcam ab5103), was performed for 2 hours at room temperature in 1% BSA PBSt at a concentration of 1:250 in a humidified chamber. Primary antibody was decanted and slides washed three times with PBS for 5 minutes each. Goat anti-Rabbit 488 secondary antibody (Invitrogen A11008) was performed for 1 hour at room temperature at a concentration of 1:500 in a humidified chamber. Secondary antibody was decanted and slides washed three times with PBS for 5 minutes each followed by VECTASHIELD Antifade Mounting Medium with DAPI (Vector Labs H-1200), sealing coverslip, and imaging on the Zeiss Axioskop 2 Plus. All studies were repeated with at least two distinct sperm samples. Exposures for different sperm preps were captured using identical light intensity and exposure times, set to enable visualization of cell-free DNA in DAPI images, and set just below saturation for cit-H3 images. Note that secondary alone controls for cit-H3 were clean (not shown).
7
Neutrophil Immunofluorescence Staining
8
Histone H3 Citrullination Immunostaining
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After sperm prep, samples were dried onto VWR® Superfrost® Plus Micro Slides (48311-703), fixed for 10 minutes in 4% paraformaldehyde solution, washed 3x with PBS, and permeabilized for 10 min with 0.1% triton in PBS. Blocking was performed with 10% BSA in PBS for 1 hour at room temperature in a humidified chamber. Staining with primary antibody, Anti-Histone H3 (citrulline R2 + R8 + R17 (Abcam ab5103), was performed for 2 hours at room temperature in 1% BSA PBSt at a concentration of 1:250 in a humidified chamber. Primary antibody was decanted and slides washed three times with PBS for 5 minutes each. Goat anti-Rabbit 488 secondary antibody (Invitrogen A11008) was performed for 1 hour at room temperature at a concentration of 1:500 in a humidified chamber. Secondary antibody was decanted and slides washed three times with PBS for 5 minutes each followed by VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Labs H-1200), sealing coverslip, and imaging on the Zeiss Axioskop 2 Plus.
9
Western Blot Analysis of Citrullinated Histone H3
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For Western blot analysis, the reaction was stopped by adding to the cell suspension an equal volume of 2x reducing Laemmli's lysis buffer, added with 2 mmol/L sodium orthovanadate, 5 mmol/L EGTA, 5 mmol/L EDTA, 10 mmol/L sodium pyrophosphate, 10 mmol/L iodoacetic acid, 1 mmol/L phenylmethylsulphonyl fluoride, 10 mmol/L sodium fluoride, 10 μg/mL leupeptin and aprotinin, 1 mg/mL trypsin/chymotrypsin inhibitor. Samples were boiled for 10 min and centrifuged for 10 min at 10,000g. Aliquots of 100 μl, corresponding to 0.2 x 10 6 neutrophil total lysate (supernatant and cells), were loaded into 10% gradient sodium dodecyl sulphate-polyacrylamide gel. Proteins were transferred onto nitrocellulose sheets and nonspecific sites blocked using 1% bovine serum albumin (BSA) in Tris-buffered saline overnight at room temperature on a horizontal shaker. The presence of citrullinated Hystone-3 was analysed by immunoblotting with a rabbit, polyclonal, anti-Histone H3 (citrulline R2+R8+R17) (ab5103Abcam, Cambridge, Mass., USA), (1:1000; 1 hour at room temperature) specific antibody, followed by incubation with anti-rabbit ECL-conjugated secondary antibody (1:5000; 1 hour at room temperature). ECL reagent (Perkin/Elmer, Inc) (MA/USA) was used for the detection of luminescence, by UVITEK.
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