OVA peptide (OVAp) which encompasses aa residues 323–339 (ISQAVHAAHAEINEAGR) of OVA is recognized by OT-II-TCR transgenic T cells in the context of H-2b MHC haplotype and was purchased from EZBiolab (Carmel, IN). OVA-Alexa 488 was purchased from Molecular Probes (Eugene, OR) and was used to track Ag uptake and DC-trafficking to the thymus.
Ova alexa488
Manufactured by Thermo Fisher Scientific
Sourced in Panama, Germany
The OVA-Alexa488 is a fluorescently-labeled protein that can be used for detection and quantification purposes in various laboratory applications. The product utilizes Alexa Fluor 488 dye to label ovalbumin, a common protein found in egg white. This allows for sensitive and specific identification of the labeled protein through fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dq ova, by Thermo Fisher Scientific (1 mentions)
Cpg c, by InvivoGen (1 mentions)
Sigma adjuvant system, by Merck Group (1 mentions)
Cd16 32 2.4g2, by BD (1 mentions)
5 bromo 2 deoxy uridine labeling and detection kit 3, by Roche (1 mentions)
Il 12p70, by BD (1 mentions)
Fitc or pe conjugated monoclonal antibodies abs, by BD (1 mentions)
Non labeled abs, by BD (1 mentions)
Recombinant mouse granulocyte macrophage colony stimulating factor gm csf, by R&D Systems (1 mentions)
Lipopolysaccharide lps, by Thermo Fisher Scientific (1 mentions)
Cd8 pe, by BD (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Collagenase d, by Roche (1 mentions)
Tnf α, by BD (1 mentions)
Ifn γ, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Interleukin 2 (il 2), by BD (1 mentions)
Pe conjugated anti cd11c, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
5 protocols using ova alexa488
1
OVA Peptide Recognition and Trafficking
2
Evaluating Antigen Uptake and Processing
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DCs were incubated at 37 °C with OVA-Alexa 488 or DQ-OVA (Molecular Probes, Inc, Eugene, OR) for 10 or 90 min respectively. Live cells with OVA uptake or OVA processing were analyzed by flow cytometry. DCs incubated at 4 °C were used as a negative control.
3
Murine Immunization Protocol with Ovalbumin
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Ovalbumin protein (OVA), Sigma Adjuvant System (SAS) and IFA were from Sigma-Aldrich, Ea (ASFEAQGALANIAVDKA), Ea-FITC, 1W1K (EAWGALANKAVDKA) from Genecust, Alum and OVA-Alexa488 from Invitrogen, NP-OVA from Biosearch Technologies Inc. CpG-A (5′-GGGGTCAACGTTGAGGGGGG-3′), CpG-B (5′-TCCATGACGTTCCTGACGTT-3), CpG-B control (5′-TCCATGAGCTTCCTGAGCTT-3′, same sequence that CpG-B but contains GpC dinucleotides instead of CpG and can therefore be used as a negative control for CpG-B), CpG-C (5′-TCGTCGTTTTCGGCGCGCGCCG-3′) were from Invivogen and carboxylated green fluorescent microspheres from Polysciences (Warrington, PA). Mice were either i.p. injected with 200 μl or immunised s.c. at the base of tail with 100 μl each side with 40 μg of peptide 1W 1K, 100 μg of OVA or NP-OVA, 200 μg of Ea-FITC or 1.2 × 1010 beads in the indicated adjuvant.
4
Murine Dendritic Cell Isolation and Stimulation
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Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (rmIL)-4 were purchased from R&D Systems (Minneapolis, MN, USA), propidium iodide (PI), and ovalbumin (OVA) were purchased from Sigma-Aldrich (Steinheim, Germany), and lipopolysaccharide (LPS) and OVA-Alexa 488 were purchased from Invitrogen (Carlsbad, CA, USA). The following FITC- or PE-conjugated monoclonal antibodies (Abs) and non-labeled Abs were purchased from BD Biosciences (San Jose, CA, USA): FITC-annexin V, CD16/32 (2.4G2), CD11c (HL3), IA[b] (AF6–120.1), IFN-γ, CD4, PE-CD8, CD4. Cytokine ELISA primary and secondary -antibodies specific for murine IL-1β, IL-6, IL-12p70, IFN-γ, IL-2, IL-10, and TNF-α were purchased from BD Biosciences (San Jose, CA, USA). 5-Bromo-2′-Deoxy-Uridine Labeling and Detection Kit III and collagenase D were purchased from Roche (Salt Lake City, UT, USA).
5
Quantifying Antigen Uptake by Bone Marrow-Derived Dendritic Cells
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Antigen uptake by BMDCs was measured using the standard method. OVA-Alexa488 (Invitrogen) was used as the model antigen. BMDCs (2×105) were incubated for various time points at 37 °C with OVA-Alexa488 (50 µg/mL) or OVA-Alexa488-MNPs (OVA-Alexa488 50 µg/mL/MNPs 25, and 50 µg/mL) particles. Uptake was terminated by washing the cells with ice-cold fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline containing 1% FBS and 0.1% NaN3). Cells were stained with PE-conjugated anti-CD11c (BD Biosciences). Alexa488 fluorescence of CD11cpositive cells was measured using an FACS CantoII (BD Biosciences). The morphology of the BMDCs was examined using a Confocal Laser Scanning Microscope (CLSM) (PE: excitation 496, emission; 578 FITC: excitation 488, emission 519).
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