14C-labeled acyl-coenzyme A (acetyl-CoA, malonyl-CoA, and succinyl-CoA) was purchased from PerkinElmer (Waltham, MA, USA). Calf thymus histone and unlabeled acyl-coenzyme A were purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blotting, the following antibodies were used: anti-IKKα (Santa Cruz, sc-7218) (CA, USA), anti-HDAC2 (ABR, PA I-861) (Golden, CO, USA), anti-acetyl histone H3 (Millipore, 06-594) (Bedford, MA, USA), anti-E1A-binding protein p300 (p300) (Santa Cruz, sc-585), and anti-CREB-binding protein (CBP) (Abcam, ab3652) (Cambridge, UK).
Calf thymus histone
Manufactured by Merck Group
Sourced in United States
Calf thymus histones are a type of laboratory equipment used for various research and experimental purposes. Histones are a class of proteins found in the nuclei of eukaryotic cells, which play a crucial role in the organization and compaction of DNA within the cell's chromatin structure. The calf thymus histones are a commonly used source of these proteins for scientific investigations and applications.
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Lab products found in correlation
Biotinylated anti mouse igm, by Jackson ImmunoResearch (3 mentions)
Alkaline phosphatase conjugated anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Biotinylated anti kappa ab, by Thermo Fisher Scientific (2 mentions)
Vector blue alkaline phosphatase substrate kit 3, by Vector Laboratories (2 mentions)
Streptavidin sa alkaline phosphatase, by Vector Laboratories (2 mentions)
Nucleosome coated plates, by Merck Group (2 mentions)
Sa alkaline phosphatase or alkaline phosphatase conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Elispot plate imaging and analysis system, by Cellular Technology (2 mentions)
Calf thymus dna, by Merck Group (2 mentions)
Tetramethylbenzidine tmb substrate, by Merck Group (2 mentions)
Hrp anti mouse igg detection antibody, by Southern Biotech (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Protac, by Sekisui (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Anti acetyl histone h3, by Merck Group (1 mentions)
Acetyl coa, by PerkinElmer (1 mentions)
Malonyl coa, by PerkinElmer (1 mentions)
Anti ikkα, by Santa Cruz Biotechnology (1 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
27 protocols using calf thymus histone
1
Histone Acetylation Assay Protocol
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14C-labeled acyl-coenzyme A (acetyl-CoA, malonyl-CoA, and succinyl-CoA) was purchased from PerkinElmer (Waltham, MA, USA). Calf thymus histone and unlabeled acyl-coenzyme A were purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blotting, the following antibodies were used: anti-IKKα (Santa Cruz, sc-7218) (CA, USA), anti-HDAC2 (ABR, PA I-861) (Golden, CO, USA), anti-acetyl histone H3 (Millipore, 06-594) (Bedford, MA, USA), anti-E1A-binding protein p300 (p300) (Santa Cruz, sc-585), and anti-CREB-binding protein (CBP) (Abcam, ab3652) (Cambridge, UK).
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14C-labeled acyl-coenzyme A (acetyl-CoA, malonyl-CoA, and succinyl-CoA) was purchased from PerkinElmer (Waltham, MA, USA). Calf thymus histone and unlabeled acyl-coenzyme A were purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blotting, the following antibodies were used: anti-IKKα (Santa Cruz, sc-7218) (CA, USA), anti-HDAC2 (ABR, PA I-861) (Golden, CO, USA), anti-acetyl histone H3 (Millipore, 06-594) (Bedford, MA, USA), anti-E1A-binding protein p300 (p300) (Santa Cruz, sc-585), and anti-CREB-binding protein (CBP) (Abcam, ab3652) (Cambridge, UK).
2
HUVEC Apoptosis Assay with APC
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Human umbilical vein endothelial cells (HUVEC) from ATCC (Manassas, VA, USA, Catalog# CRL-1730) were maintained in ATCC-formulated F-12K Medium (Catalog# 30-2004) supplemented with 10% fetal bovine serum. Cells were seeded at 2 × 106 cells per mL 1 d before the experiment. Confluent monolayers of HUVECs were incubated with 20 nM APC in the presence of mAb (0, 3, 30, and 300 nM). Apoptosis was induced by incubation with calf thymus histone (Sigma-Aldrich) at 2 μM for 4 h. HUVECs were detached from the plate by rinsing once with PBS, and incubation with Enzyme-free Cell Dissociation Buffer (Gibco, Catalog#13151014) for 10 min followed by collection of the cells using a cell scraper, and cell viability was assessed using dyes, propidium iodide, or 7-AAD, as detected by fluorescence-activated cell-sorting analysis. The results are shown as mean (SD) of 3 independent experiments (P < 0.01 [ANOVA]).
3
ELISpot Assay for Antibody-Producing Cells
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ELISpot assays were performed as described (Wong et al., 2012 (link)). Briefly, splenocytes in RPMI containing 10% fetal bovine serum were plated at a concentration of 1 × 105 cells/well onto anti-IgM-, anti-IgG-, salmon sperm dsDNA- (Invitrogen, Grand Island, NY), calf thymus histone- (Sigma Aldrich, St. Louis, MO), or nucleosome-coated plates (Millipore, Bedford, MA). Serially diluted (1:2) cells were incubated for 6 hr at 37°C. IgM-producing AFCs were detected using biotinylated anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA) and streptavidin (SA)-alkaline phosphatase (Vector Laboratories, Burlingame, CA). IgG-producing AFCs were detected using alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes, Grand Island, NY). dsDNA-, histone-, and nucleosome-specific AFCs were detected by biotinylated anti-kappa Ab (Invitrogen, Grand Island, NY) and SA-alkaline phosphatase or alkaline phosphatase-conjugated anti-mouse IgG (Molecular Probes, Grand Island, NY). Plates were developed using the Vector Blue Alkaline phosphatase Substrate Kit III (Vector Laboratories, Burlingame, CA). ELISpots were enumerated using a computerized ELISpot plate imaging and analysis system (Cellular Technology, Shaker Heights, OH).
4
Enumeration of Antigen-Specific Antibody-Producing Cells
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In brief, splenocytes in RPMI containing 10% FBS were plated at a concentration of 106 cells per well onto anti-IgM–, anti-IgG–, salmon sperm dsDNA– (Invitrogen), calf thymus histone– (Sigma-Aldrich), or nucleosome- (histone plated on a layer of dsDNA coating) coated multiscreen 96-well filtration plates (EMD Millipore). Serially diluted (1:2) cells were incubated for 6 h at 37°C. IgM-producing AFCs were detected using biotinylated anti–mouse IgM (Jackson ImmunoResearch Laboratories, Inc.) and streptavidin–alkaline phosphatase (Vector Laboratories). IgG-producing AFCs were detected using alkaline phosphatase–conjugated anti–mouse IgG (Molecular Probes). dsDNA-, histone-, and nucleosome-specific AFCs were detected by biotinylated anti-κ Abs (Invitrogen) followed by streptavidin–alkaline phosphatase (Vector Laboratories) or alkaline phosphatase–conjugated anti–mouse IgG (Molecular Probes). Plates were developed using a blue alkaline phosphatase substrate kit (III; Vector Laboratories). ELISPOTs were enumerated and analyzed using a computerized ELISPOT plate imaging/analysis system (Cellular Technology).
5
Histone-Induced Endothelial Cell Responses
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Pooled human umbilical vein endothelial cells (HUVECs) from 5 individual female donors were purchased from Lonza (Barcelona, Spain) and were grown in Medium 199 (Sigma-Aldrich, Madrid, Spain) supplemented with 20% fetal bovine serum (Gibco, Invitrogen, Barcelona, Spain), endothelial cell growth supplement from bovine neural tissue (ECGS, Sigma-Aldrich), and heparin sodium salt from porcine intestinal mucosa (Sigma-Aldrich). Cells were routinely grown in an incubator at 37 °C with 5% CO2. HUVECs from passages 3 to 5 were used in this study. When they reached confluence, the media was changed and cells underwent 4 h exposure to different calf thymus histone concentrations (Sigma-Aldrich, St. Louis, MO, USA): 10, 25, 50, or 100 µg/mL prepared in PBS. In some experiments, 30 µM apocynin (Sigma-Aldrich), 10 µM indomethacin (Sigma-Aldrich), 10 µM celecoxib (Sigma-Aldrich), 100 µmol/l tempol (Sigma-Aldrich), 20 µM Bay11-7082 ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile; Sigma-Aldrich), 20 µg/mL oxPAPC (Invivogen, Toulouse, France), 0.7 µM iODN (inhibitory oligodeoxynucleotide with phosphorothioate backbone, Enzo Life Science, Farmingdale, USA), and 3 µM CLI-095 (Invivogen) were added to HUVEC 1 h prior to histone treatment.
6
Autoantigen Reactivity ELISA Protocol
7
Autoantibody Detection in Mouse Serum
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At the indicated time points, mice were bled and serum isolated using Microtubes Z-gel (Sarstedt). Nunc Maxisorp plates were coated overnight at 4°C using PBS solutions of: 100 μg/mL salmon sperm DNA (Thermo Fisher Scientific), 10 μg/mL calf thymus histone (Sigma Aldrich), 1 U/well ribosomal P (Immunovision, Springdale, AK, USA), 1 U/well Smith antigen (Immunovision) or 20 μg/mL DWEYS peptide (Bachem, Bubendorf, Switzerland). DWEYS peptide coating supported detection of antibodies against NMDAR.
Plates were washed, blocked, prior to the addition of mouse serum dilutions of 1:100 or 1:300 in PBS/1% BSA for 2 h. Washed plates were incubated with HRP-conjugated anti-IgG-specific detection antibodies for 2 h (1:10′000). Washed plates were incubated with 100 μL/well of TMB substrate (BD Biosciences) and the reaction stopped by addition of 1 N HCl. The OD (450 nm) of samples was measured using a Spectramax M5 (Molecular Devices) and data expressed with background subtracted. Ro60 immunoglobulin titers were determined in 1:100 serum dilutions using an ELISA kit (Alpha Diagnostic, San Antonio, TX, USA) and reported as U/ml.
Plates were washed, blocked, prior to the addition of mouse serum dilutions of 1:100 or 1:300 in PBS/1% BSA for 2 h. Washed plates were incubated with HRP-conjugated anti-IgG-specific detection antibodies for 2 h (1:10′000). Washed plates were incubated with 100 μL/well of TMB substrate (BD Biosciences) and the reaction stopped by addition of 1 N HCl. The OD (450 nm) of samples was measured using a Spectramax M5 (Molecular Devices) and data expressed with background subtracted. Ro60 immunoglobulin titers were determined in 1:100 serum dilutions using an ELISA kit (Alpha Diagnostic, San Antonio, TX, USA) and reported as U/ml.
8
Binding Assays for G-Protein Coupled Receptors
9
ELISpot Assay for Antibody-Producing Cells
10
Histone-induced Acute Lung Injury
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All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Nagoya University School of Medicine (approval number: 20353) and performed in accordance with the relevant guidelines and regulations, including the ARRIVE guidelines. Nine to twelve‐week‐old male Mac‐1 knockout (Mac‐1−/−) C57BL/6 mice were obtained from T. N. Mayadas, and C57BL/6 WT mice were purchased from Japan SLC (Shizuoka, Japan). All mice were maintained in virus‐ and antibody‐free facilities and given free food and water access. The histone‐induced ALI model was constructed as previously described [22 ]. Briefly, WT and Mac‐1−/−mice received a single tail vein injection of calf thymus histone which contains histone H3, H4, H2A, and H2B (60 μg·g−1; Sigma‐Aldrich, St Louis, MO, USA) and were monitored for up to 1 h for survival time analysis. The survival analysis was independently conducted before other experiments.
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