B cells were isolated using CD19 Microbeads (Miltenyi Biotec) and cultured at 2.5×106 cells/ml for 48h in the presence of soluble recombinant human CD40L (100ng/ml) (MegaCD40L, Enzo LifeSciences) or PBS as control before cells were lysed using ice cold IP Lysis Buffer (Pierce) in the presence of protease inhibitors. 500 µg protein lysate was used for each IP using the Immunoprecipitation Kit Dynabeads® Protein G (Life Technologies). Briefly, 5 µg mAb (CD45, clone F10–89-4) or isotype control (mouse IgG2a, BioRad) was bound to 50 µL Dynabeads and crosslinked using BS3 reagent (ThermoFisher) according to manufacturers instructions, before cell lysate was added at 4°C for 4h. Beads were washed and bound protein was eluted and sample buffer (NuPAGE LDS sample buffer) and DTT was added. Samples were heated to 70°C for 10 min before loaded onto SDS-PAGE. Gels were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked in 5% nonfat dry milk and 0.1% Tween. The membrane was probed with goat anti-Galectin-1 polyclonal antibody (1:2000)(R&D Systems) and anti-goat IgG HRP (1:1000)(R&D Systems) or anti-CD45 (1:1000) and anti-mouse IgG HRP (1:1000) (R&D Systems).
Anti cd45
Manufactured by R&D Systems
Sourced in United States
Anti-CD45 is a laboratory antibody product used to detect and identify CD45-positive cells in flow cytometry and other immunoassay applications. CD45 is a transmembrane protein tyrosine phosphatase expressed on the surface of most hematopoietic cells.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Mouse igg2a, by Bio-Rad (2 mentions)
Bs3 reagent, by Thermo Fisher Scientific (2 mentions)
Goat anti galectin 1 polyclonal antibody, by R&D Systems (2 mentions)
Anti mouse igg hrp, by R&D Systems (2 mentions)
Cd19 microbeads, by Miltenyi Biotec (2 mentions)
Megacd40l, by Enzo Life Sciences (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Biotinylated anti rat igg, by Vector Laboratories (1 mentions)
Streptavidin 488, by Vector Laboratories (1 mentions)
M o m immunodetection kit, by Vector Laboratories (1 mentions)
Biorevo bz 9000, by Keyence (1 mentions)
Anti cd115, by Thermo Fisher Scientific (1 mentions)
Anti ccr2, by R&D Systems (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Anti cd146, by R&D Systems (1 mentions)
Anti cd105, by R&D Systems (1 mentions)
Anti cd90, by R&D Systems (1 mentions)
9 protocols using anti cd45
1
Galectin-1 Immunoprecipitation from CD40L-stimulated B Cells
2
Assessing Renal Fibrosis in UUO Mice
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To evaluate the infiltration/accumulation of bone marrow-derived fibrotic cells in the kidneys of the UUO mice model, double staining of CD45 and αSMA was performed using of formalin-fixed paraffin-embedded kidney tissues. For αSMA staining, the M.O.M. Immunodetection Kit (Vector Laboratories) was used as per the manufacturer’s instructions. Anti-CD45 (R&D systems) was used, followed by biotinylated anti-Rat IgG (Vector Laboratories) and streptavidin 488 (Vector Laboratories). After mounting with Vectashield with DAPI (Vector Laboratories), the sample was observed under a fluorescence microscope (Biorevo BZ-9000, Keyence).
3
Flow cytometric analysis of myeloid cells
4
Phenotypic Characterization of MSCs
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MSCs were incubated with anti-CD19, anti-CD146, anti-CD44, anti-CD45, anti-CD90, and anti-CD105 antibodies (R&D, USA) at room temperature for 30 min. After washing twice with PBS, the MSCs were incubated with a FITC-labeled secondary antibody in the dark for 30 min. After washing, the cells were suspended in PBS and analyzed on a flow cytometer.
5
Lymphocyte Immunophenotyping and Pneumococcal Vaccine Response
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B and T lymphocyte immunophenotyping was completed from fresh heparin-blood samples. Four or 10-color flow cytometry panel with monoclonal antibodies (mAb) against the surface antigens IgM, IgD, CD3, CD4, CD8, CD16҄56, CD19, CD21, CD27, CD33, CD34, CD38, CD45, CD56, CD57, CD133, HLA-DR, CD62L, CD45RA and CD45RO (BD Biosciences). T cell immunophenotyping was studied with the antibody panel including anti-CD45, -CD3, -CD4, -CD8, -CD45RA, and -CCR7 (R&D Systems). B and T cell phenotyping was completed as previously described and B and T cell subpopulations were analyzed according to published protocols (25 (link)–27 (link)). The vaccine response to pneumococcal polysaccharide vaccine (Pneumovax®) was performed as described (28 (link)).
6
Quantifying Demyelination and Axonal Loss
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Inflammatory demyelination was measured by staining for myelin basic protein (MBP) with anti‐MBP antibody (1:200, BioRad) and axonal loss was assessed using anti‐SMI‐31 (1:1000, Bio‐Legend). The inflammatory activity was analyzed using anti–CD45 (1:100, R&D systems) anti‐CD4. We used anti‐glial fibrillary acidic protein (GFAP, 1:500, Dako) for the detection of astrocytes. All primary antibodies were detected with appropriate conjugated secondary antibody (1:500, Thermofisher, UK) according to established standard protocols. Classic hematoxylin and eosin staining was used to analyze inflammatory activity. In brief, sections were stained with hematoxylin for 5 min, rinsed with distilled water then incubated in 1% acid ethanol for 10 s and rinsed sufficiently. Eosin was then applied for 5 min and rinsed sufficiently. Sections were dehydrated in gradient ethanol solutions and transparentized with xylene before being embedded with DPX mounting media.
7
Comprehensive Leukocyte Immunophenotyping Protocol
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Lymphocyte, B-, and T-cell phenotyping were performed as previously published69 (link)–71 (link) as a service at Helsinki University Hospital (analysis codes: 6258smB-Fc, 21388Tdif-Fc, and 8302LyDiff-T). Briefly, for flow cytometry, cells in EDTA samples were stained using whole blood technique, and analyzed on a FACSCanto II flow cytometer (Beckton Dickinson Biosciences, San Jose, CA, USA). Fresh heparin-blood samples or peripheral blood mononuclear cells (PBMCs) were used for B- and T-lymphocyte immunophenotyping. Four- or 10-color flow cytometry panel with monoclonal antibodies (mAbs) against the surface antigens IgM, IgD, CD3, CD4, CD8, CD16⁄56, CD19, CD21, CD27, CD33, CD34, CD38, CD45, CD56, CD57, CD133, HLA-DR, CD62L, CD45RA, and CD45RO (BD Biosciences, San Jose, CA)70 (link). The memory status of T cells was studied with the antibody panel including anti-CD45, -CD3, -CD4, -CD8 -CD45RA, and -CCR7 (R&D Systems, Minneapolis, MN, USA)70 (link),71 (link).
8
Galectin-1 Immunoprecipitation from CD40L-stimulated B Cells
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B cells were isolated using CD19 Microbeads (Miltenyi Biotec) and cultured at 2.5×106 cells/ml for 48h in the presence of soluble recombinant human CD40L (100ng/ml) (MegaCD40L, Enzo LifeSciences) or PBS as control before cells were lysed using ice cold IP Lysis Buffer (Pierce) in the presence of protease inhibitors. 500 µg protein lysate was used for each IP using the Immunoprecipitation Kit Dynabeads® Protein G (Life Technologies). Briefly, 5 µg mAb (CD45, clone F10–89-4) or isotype control (mouse IgG2a, BioRad) was bound to 50 µL Dynabeads and crosslinked using BS3 reagent (ThermoFisher) according to manufacturers instructions, before cell lysate was added at 4°C for 4h. Beads were washed and bound protein was eluted and sample buffer (NuPAGE LDS sample buffer) and DTT was added. Samples were heated to 70°C for 10 min before loaded onto SDS-PAGE. Gels were transferred to polyvinylidene difluoride membrane (Bio-Rad) and blocked in 5% nonfat dry milk and 0.1% Tween. The membrane was probed with goat anti-Galectin-1 polyclonal antibody (1:2000)(R&D Systems) and anti-goat IgG HRP (1:1000)(R&D Systems) or anti-CD45 (1:1000) and anti-mouse IgG HRP (1:1000) (R&D Systems).
9
Magnetic Separation of White Blood Cells
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WBCs were collected from a whole blood sample after red blood cell lysis and were magnetically labeled with superparamagnetic nanoparticles (Ademtech SA, Pessac, France, MasterBeads Carboxylic Acid 0215). The nanoparticles were 500 nm in diameter and composed of a magnetic core (approximately 70% iron oxide) encapsulated by a hydrophilic polymer shell with carboxyl groups on its surface. They were functionalized with both anti-CD45 and anti-CD15 antibodies (purchased from R & D Systems, Minneapolis, MO, USA) to enhance WBC magnetic labeling. Finally, WBCs were suspended in 500 µL of EDTA (2 mM, PBS-BSA 2%) at a concentration of 6.105 WBCs/mL. PC-3 cancer cells were spiked into the white blood cell sample, at a concentration of 4.104 PC-3/mL, to study the specificity of the trapping. WBCs were stained with Hoechst 33342 (Ready Flow Reagent™, ThermoFisher Scientific, Waltham, MA, USA) and PC-3 cells were tracked with a green dye (CellTracker™ Green CMFDA Dye, ThermoFisher Scientific, Waltham, MA, USA).
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