Anti cd11c alexa700, by Thermo Fisher Scientific - Product details

1

Colon and Caecum Immune Cell Isolation

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Caecum and colon were harvested at autopsy and digested in RPMI containing 5% L-glutamine, 5% penicillin streptomycin, 10% fetal bovine serum (Sigma Aldrich, Dorset, UK), collagenase (1mg/ml), and dispase (0.5mgs/ml, both Gibco, Paisley, UK) for 2 hours at 37 °C. Cells were then forced through a 70μm cell strainer, centrifuged at 405g for 5 minutes and resuspended in 10mls 80% Percoll (GE Healthcare, Buckinghamshire, UK) solution which was then overlaid on a 40% Percoll solution. Cells were centrifuged for 25 minutes at 1000g and the cells at the gradient interface harvested. Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson, Oxford, UK), anti-CD103 PE (1 μg/ml), anti-CD11c Alexa700 (2.5 μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-F4/80 APC (1 μg/ml) (all E bioscience, Hatfield, UK) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).

Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.

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2

Dendritic Cell Isolation and Stimulation

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Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×10⁶ cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).

Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.

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3

Colon and Caecum Immune Cell Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Caecum and colon were harvested at autopsy and digested in RPMI containing 5% L-glutamine, 5% penicillin streptomycin, 10% fetal bovine serum (Sigma Aldrich, Dorset, UK), collagenase (1mg/ml), and dispase (0.5mgs/ml, both Gibco, Paisley, UK) for 2 hours at 37 °C. Cells were then forced through a 70μm cell strainer, centrifuged at 405g for 5 minutes and resuspended in 10mls 80% Percoll (GE Healthcare, Buckinghamshire, UK) solution which was then overlaid on a 40% Percoll solution. Cells were centrifuged for 25 minutes at 1000g and the cells at the gradient interface harvested. Fc receptors were blocked using anti-CD16/32 (2 μg/ml E bioscience, Hatfield, UK). Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson, Oxford, UK), anti-CD103 PE (1 μg/ml), anti-CD11c Alexa700 (2.5 μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-F4/80 APC (1 μg/ml) (all E bioscience, Hatfield, UK) and acquired by flow cytometry on the BD LSRII. Data was analysed using FlowJo flow cytometry software (Tree Star inc. Oregon, US).

Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.

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4

Comprehensive PBMC Immunophenotyping Protocol

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Peripheral blood mononuclear cells (PBMCs) were stained in four panels containing anti-CD3-PacificBlue (PacBlue) (antibodies from BD Biosciences unless otherwise indicated), anti-CD3-Alexa700, anti-CD25-PE-Cy7, anti-CD38-PE, anti-HLA-DR-PE-Cy7, anti-CCR5-APC, anti-CD123-PerCP-Cy5.5, anti-CD16-PacBlue, anti-CD80-FITC, anti-CD83-PE, anti-CD86-APC, anti-PD1-FITC, anti-PD-L1-PE, anti-HLA class I-APC, anti-CD69-APC-Cy7; anti-CD4-Qdot655, anti-CD8-PE-Cy5.5, anti-CD14-Qdot605 (Invitrogen); anti-CD45RA-ECD, anti-CD127-PE, anti-HLA-DR-ECD, anti-CD20-ECD (Beckman Coulter); anti-CD11c-Alexa700 (eBioscience); and anti-CD27-APC-Cy7 (BioLegend). A staining reagent for dead cells (Invitrogen Aqua Live/Dead Fixable Stain) was included. Cells were then washed and fixed in PBS containing 1% paraformaldehyde or permeabilized using a FOXP3 Fix/Perm kit (BioLegend) according to the manufacturer's instructions, intracellularly stained with anti-Ki67-Alexa 488 (BD Biosciences), anti-FOXP3-PacBlue, anti-T-Bet-BV711 (BioLegend), and anti-Eomes-eFluor 660 (eBioscience), fixed, and analyzed with a LSR II cytometer (Becton Dickinson) and FlowJo.

Méndez-Lagares G., Lu D., Chen C., Terrault N., Segal M.R., Khalili M., Monto A., Shen H., Manos M.M., Lanier L.L., Ryan J.C., McCune J.M, & Hartigan-O'Connor D. (2017). Memory T-cell proliferation before HCV therapy predicts antiviral immune responses and treatment success. Journal of immunology (Baltimore, Md. : 1950), 200(3), 1124-1132.

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5

Dendritic Cell Isolation and Stimulation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Bone marrow was harvested and cultured as previously described[23 (link)]. On day six of the culture the cells were harvested from the plates and semi-adherent cells removed. The cells were spun at 400g and re-suspended at a concentration of 1×10⁶ cells/ml in DC medium (RPMI 1640 supplemented with 10% LPS-free FBS (Gibco, Paisley, UK) 1% Penicillin/streptomycin and 50mM Beta-mercaptoethanol (Sigma Aldrich, Dorset UK)). The cells were stimulated overnight with LPS (100ng/ml) or T. muris antigen (5μg/ml). After 24 hours the cells were harvested and prepared for flow cytometry. FcR were blocked by incubating cells in anti-CD16/32 (2μg/ml) for 15 minutes. Cells were washed and stained with anti-CD45 PeCy7 (1 μg/ml, Becton Dickinson), anti-CD11c Alexa700 (1μg/ml), anti-MHC-II FITC (2.5 μg/ml) and anti-CD86 PE (1 μg/ml) (all E Bioscience) and acquired using a LSRII flow cytometer (Becton Dickinson Biosciences, Oxfordshire). Data was analysed using Flow Jo flow cytometry analysis software (Tree Star, Inc, Oregon, US).

Bowcutt R., Bramhall M., Logunova L., Wilson J., Booth C., Carding S.R., Grencis R, & Cruickshank S. (2014). A role for the pattern recognition receptor Nod2 in promoting recruitment of CD103+ Dendritic Cells to the colon in response to Trichuris muris infection. Mucosal immunology, 7(5), 1094-1105.

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Anti cd11c alexa700

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5 protocols using anti cd11c alexa700

Colon and Caecum Immune Cell Isolation

Dendritic Cell Isolation and Stimulation

Colon and Caecum Immune Cell Isolation

Comprehensive PBMC Immunophenotyping Protocol

Dendritic Cell Isolation and Stimulation

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