Multiscreen cellulose filter plates (Millipore) were coated overnight with 10 μg of histone from calf thymus (Sigma-Aldrich). Cells from the mLN were collected and plated starting at 3 × 106/well and in 2-fold serial dilutions in complete RPMI 1640 containing 10% FBS. After 5 h, the wells were washed with PBS containing 0.5% BSA and 0.05% Tween 20, and IgG was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson immunoresearch). Spots were counted using a dissecting microscope. Images were taken using a CTL-immunoSpot S5 analyzer.
Histone from calf thymus
Manufactured by Merck Group
Sourced in Germany
Histone from calf thymus is a type of nuclear protein found in eukaryotic cells. It plays a fundamental role in the structure of chromatin by facilitating the packaging of DNA within the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Alkaline phosphatase conjugated goat anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Multiscreen cellulose filter plates, by Merck Group (2 mentions)
Spermine, by Merck Group (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Urea, by Merck Group (1 mentions)
Citric acid monohydrate, by Merck Group (1 mentions)
Fructose, by Merck Group (1 mentions)
Zinc sulfate heptahydrate, by Merck Group (1 mentions)
Origin 7, by OriginLab (1 mentions)
C18 cartridge, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti rabbit igg, by Merck Group (1 mentions)
Anti vinculin, by Merck Group (1 mentions)
6 protocols using histone from calf thymus
1
Histone-Specific Antibody Secretion Assay
2
CACO-2 Cell Barrier Modulation by Seminal Plasma
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Nine-day post-confluent CACO-2 cell layers on Millipore PCF filters were exposed to varying concentrations of specific seminal plasma components in culture medium in the basal-lateral fluid compartment 24 hours prior to measurements of transepithelial electrical resistance (Rt) and 14C-D-mannitol permeability (Jm). Human recombinant proteins, TNF-α and IL1β, were obtained from PeproTech (Rocky Hill, NJ). IFN-γ was a product of Life Technologies (Frederick, MD). Zinc sulfate heptahydrate, histone from calf thymus, spermine, citric acid monohydrate, urea and fructose were products of Sigma-Aldrich (St. Louis, Mo.). Human recombinant EGF and TGF were products of Enzo Biochem (New York, NY) and PeproTech, respectively.
Whereas CACO-2 cultures 9 days post-seeding are not fully differentiated, barrier function is in fact near maximal.[18 (link)] Given our current study’s focus on exposure of the basal-lateral cell surface to SP, we are in fact modeling the effect of SP on an already compromised epithelium, one that allows SP penetration from the lumen into the interstitium, as would occur in an epithelium wounded mechanically or by proinflammatory cytokine damage. The epithelium in the immediate vicinity of that site of compromise would very likely not be in a completely differentiated state.
Whereas CACO-2 cultures 9 days post-seeding are not fully differentiated, barrier function is in fact near maximal.[18 (link)] Given our current study’s focus on exposure of the basal-lateral cell surface to SP, we are in fact modeling the effect of SP on an already compromised epithelium, one that allows SP penetration from the lumen into the interstitium, as would occur in an epithelium wounded mechanically or by proinflammatory cytokine damage. The epithelium in the immediate vicinity of that site of compromise would very likely not be in a completely differentiated state.
3
Histone Methylation Inhibition Assay
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For histone methylation
inhibitions, the assays were performed in 20 μL reactions containing
4.6 mM [methyl-3H]-AdoMet, 50 μg/mL histone from
calf thymus (Sigma), 12 μg/mL (0.3 μM) PRMT1 or 10 μg/mL
(0.17 μM) GLP, 100 mM KCl, 5 mM dithiothreitol (DTT), and 50
mM Tris-HCl, pH 8.5. Enzymes were preincubated with AdoMet and various
concentrations of inhibitors for 5 min at 37 °C (for PRMT1) or
30 °C (for GLP) before the addition of histone substrates. After
incubation (6.5 min for PRMT1 or 5 min for GLP), the reactions were
terminated by the addition of 20% trichloroacetic acid (TCA, Fisher
Scientific). The reaction mixtures were spotted on GF/A paper circles
(Whatman) and washed three times with 3 mL of 10% TCA and once with
3 mL of ethanol. The dried circles were subjected to liquid scintillation
counting with Cytoscint scintillant. All curves were fit individually
using Origin 7.5 software (OriginLab).
inhibitions, the assays were performed in 20 μL reactions containing
4.6 mM [methyl-3H]-AdoMet, 50 μg/mL histone from
calf thymus (Sigma), 12 μg/mL (0.3 μM) PRMT1 or 10 μg/mL
(0.17 μM) GLP, 100 mM KCl, 5 mM dithiothreitol (DTT), and 50
mM Tris-HCl, pH 8.5. Enzymes were preincubated with AdoMet and various
concentrations of inhibitors for 5 min at 37 °C (for PRMT1) or
30 °C (for GLP) before the addition of histone substrates. After
incubation (6.5 min for PRMT1 or 5 min for GLP), the reactions were
terminated by the addition of 20% trichloroacetic acid (TCA, Fisher
Scientific). The reaction mixtures were spotted on GF/A paper circles
(Whatman) and washed three times with 3 mL of 10% TCA and once with
3 mL of ethanol. The dried circles were subjected to liquid scintillation
counting with Cytoscint scintillant. All curves were fit individually
using Origin 7.5 software (OriginLab).
4
Lipopolysaccharide-free Colominic Acid and Histone Experiments
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For cell culture experiments, LPS was removed from colominic acid (Gerbu, Heidelberg, Germany) using C18 cartridges (Thermo Fisher Scientific, Dreieich, Germany), according to manufacturer specifications, as already mentioned in [33 (link)]. Used histones were: histone from calf thymus (Sigma-Aldrich, Steinheim, Germany) and recombinant human H1, H2A, H2B, H3.1, and H4 (New England Biolabs, Frankfurt am Main, Germany). All of the reagents used were of analytical grade.
5
Purification and Antibody Validation Protocol
6
Histone-Specific Antibody Secretion Assay
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