Ly6c fitc al 21

Manufactured by BD

Ly6C-FITC (AL-21) is a fluorescently-labeled antibody that binds to the Ly6C antigen. Ly6C is a cell surface marker expressed on various immune cell types. The FITC fluorescent label allows for the detection and analysis of Ly6C-positive cells using flow cytometry or other fluorescence-based techniques.

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Lab products found in correlation

Cd11c pe or bv711 hl3, by BioLegend (2 mentions) Celesta, by BD (1 mentions) F4 80 pe bm8, by BioLegend (1 mentions) Mhcii bv421 m5 114, by BioLegend (1 mentions) Cd45 bv510 30 f11, by BioLegend (1 mentions) Cd11b apc m1 70, by Thermo Fisher Scientific (1 mentions) Siglecf pe cf594, by BD (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Ly6g pe cy7, by BioLegend (1 mentions) Fixable viability dye apc efluor780, by Thermo Fisher Scientific (1 mentions) Cell strainer, by BD (1 mentions) Aqua dead live amcyan, by BD (1 mentions) Cd45.1 pe, by Thermo Fisher Scientific (1 mentions) Cd11c bv711, by BioLegend (1 mentions)

4 protocols using ly6c fitc al 21

Comprehensive Flow Cytometry Analysis of Immune Cells

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Colonic LP samples (2 million cells/staining) were stained at 4°C for 20 min with the following antibodies: Ly6C-FITC (AL-21, BD Pharmingen), F4/80-PE (BM8, BioLegend), Siglec F-PE-CF594 (E50-2440, BD Bioscience), Ly6G PE-Cy7 (1A8, BioLegend), CD11b-APC (M1/70, eBioscience), CD11c-AF700 or -APC-R700 (N418, BioLegend), MHCII-BV421 (M5/114.15.2, BioLegend), and CD45-BV510 (30-F11, BioLegend) at 4°C for 20 min. Dead cells were identified by staining with eFluor 780 Fixable Viability dye (eBioscience) and gatings were performed as described previously (22 (link)).
For analyses of integrin receptors on human monocytes, mouse Hoxb8 monocytes and mouse BMDMs, antibodies to integrins β1 (mouse 9EG7 (23 (link))/human P5D5) (24 (link)), α6 (GoH3, BD Pharmingen), α5 (mouse 5H10-27, BD Pharmingen/human P1D6) (24 (link)), α4 (PS/2, Abcam), α3 (mouse polyclonal, R&D/human P1B5) (24 (link)), β3 (mouse 2C9.G2, Biolegend/human B3A, Chemicon), and β2 (mouse C71/16, BD Pharmingen/human TS1/18, Thermo Scientific) were employed. Cells were analyzed with a Gallios (Beckman Coulter) or Celesta (BD) flow cytometer and FlowJo software.

Li L., Song J., Chuquisana O., Hannocks M.J., Loismann S., Vogl T., Roth J., Hallmann R, & Sorokin L. (2020). Endothelial Basement Membrane Laminins as an Environmental Cue in Monocyte Differentiation to Macrophages. Frontiers in Immunology, 11, 584229.

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Multicolor Flow Cytometry Immunophenotyping

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The following antibodies were used to identify cell subsets: Ly6C-FITC (AL-21, BD Biosciences), F4/80-PerCP-Cy5.5 or APC eFluor660 (BM8, eBioscience), Siglec-F-PE (E50-2440, BD Biosciences), CD11c-PE or BV711 (HL3, Biolegend), and Ly6G-PerCP eFluor710 or V450 (1A8, eBioscience or BD Biosciences). Dead cells were excluded from analyses using Fixable Viability Dye APC eFluor780 or BV506 (eBioscience).
Surface staining: Cells from all samples were adjusted to an equal concentration, and treated with anti-CD16/CD32 Fc receptor blocking antibody (clone 2.4G2) in 1x PBS (1% FBS) for 10 minutes on ice. Surface staining antibodies were then added and incubated for 15 minutes on ice. Cells were washed with 1x PBS then incubated with Fixable Viability Dye diluted in 1x PBS for 15 minutes on ice. Cells were washed, then fixed with 1% paraformaldehyde for 15 minutes on ice.
Samples were acquired using an Attune NxT Acoustic Focusing Cytometer with Attune Software or a BD FACSAria with FACSDiva Software. Analyses were performed using FlowJo v10 software (Tree Star, Inc.). Gate placement was determined using isotype, FMO, or unstained control samples. Total cell numbers of each cell subset was obtained by using the total cell counts of the compartment as described above, and multiplying by the percent of total viable cells as determined by flow cytometry.

Crane M.J., Xu Y., Henry WL J.r., Gillis S.P., Albina J.E, & Jamieson A.M. (2018). Pulmonary influenza A virus infection leads to suppression of the innate immune response to dermal injury. PLoS Pathogens, 14(8), e1007212.

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Analyzing Peritoneal and Splenic Immune Cells

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For IP injection of PITs, 2 × 10⁶ CellTracker Orange–labeled WT BMM were treated with 3 µg/ml FlaTox and IP injected into WT mice. Peritoneal fluid was collected at 2 h after IP injection and stained. For isolation of splenocytes, the spleen were treated with Collagenase D, RBC lysis buffer, and flushed through a cell strainer (Falcon). Cells were Fc-blocked with anti-CD16/CD32 and stained with anti–F4-80-PacBlue (BM8), CD11b-APC (M1/70), Ly6G-APC Cy7 (1A8), CD68-PE (FA-11) and Ly6C-FITC (AL-21) and cell viability marker Aqua dead live-AmCyan (all from BD), fixed, and analyzed on a LRSIII (BD; UNC Flow Cytometry Core Facility).

Jorgensen I., Zhang Y., Krantz B.A, & Miao E.A. (2016). Pyroptosis triggers pore-induced intracellular traps (PITs) that capture bacteria and lead to their clearance by efferocytosis. The Journal of Experimental Medicine, 213(10), 2113-2128.

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Multiparametric Immune Cell Profiling

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The following antibodies were used to identify cell subsets: Ly6C-FITC (AL-21, BD Biosciences), F4/80-APC eFluor660 (BM8, eBioscience), Siglec-F-PE or AR700 (E50-2440, BD Biosciences), CD11c-PE or BV711 (HL3, BioLegend), Ly6G-PerCP eFluor710 or V450 (1A8, eBioscience or BD Biosciences), CD45.2-APC/Fire750 or V450 (104, BioLegend or eBioscience), CD45.1-PE (A20, eBioscience), and CD11c-BV711 (N418, BioLegend). Dead cells were excluded from analyses using Fixable Viability Dye APC BV506 (eBioscience).
Surface staining: Cells were treated with anti-CD16/CD32 Fc receptor blocking antibody (clone 2.4G2) in 1x PBS (1% FBS) for 10 minutes on ice. Cells were then centrifuged and resuspended in 1x PBS (1% FBS) containing antibodies and incubated for 15 minutes on ice. Cells were washed with 1x PBS then incubated with Fixable Viability Dye diluted in 1x PBS for 15 minutes on ice.
Cells were washed, then fixed with 1% paraformaldehyde for 15 minutes on ice. Samples were acquired using an Attune NxT Acoustic Focusing Cytometer with Attune Software.
Analyses were performed using FlowJo v10 software (Tree Star, Inc.). Gate placement was determined using fluorescence minus one and unstained control samples.

Crane M.J., Xu Y., Monaghan S.F., Hall B.M., Albina J.E., Henry W.P., Tran H.L., Chhabria K., Jordon A.R.D., Carlsen L., & Jamieson A.M. (2020). Pulmonary infection interrupts acute cutaneous wound healing through disruption of chemokine signals.

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