CD184 (CXCR4) Monoclonal Antibody (2B11), Alexa Fluor 488, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
BALB/c spleens from naïve and tumor mice were mechanically digested and placed through a 70-μm cell strainer to obtain single cell suspensions. To avoid nonspecific staining, single cells Naïve and suture-inflamed BALB/c corneas were harvested and immersed in PBS containing 20 mM EDTA (Sigma-Aldrich) at 37°C for 30 minutes, and the corneal epithelium was removed with forceps to allow investigation of the corneal epithelium and stroma separately. Following two washes with PBS, the corneal stroma was cut into pieces and digested with 2 mg/mL collagenase D (Roche, Indianapolis, IN, USA) and 2 mg/mL DNAse I (Sigma-Aldrich, St. Louis, MO, USA) in DMEM for 60 minutes at 37°C. After digestion, corneal single cell suspensions were passed through a 70-μm cell strainer, and corneal epithelia and stroma were pooled into one and two separate samples per state, respectively, and blocked with Fc-block (as above).	Samples were stained with fluorophore-conjugated antibodies against cell surface markers CXCR4. Single cell suspensions were then washed, fixed, and permeabilized (Cat. 555028; BD Bioscience)	Single cell suspensions were then stained with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), and following a final wash underwent flow cytometric analysis using BD LSR II Cytometer (BD Biosciences). Acquired data were analyzed by FlowJo v10 (FlowJo, LLC, Ashland, OR, USA).

Upstream tips
BALB/c spleens from naïve and tumor mice were mechanically digested and placed through a 70-μm cell strainer to obtain single cell suspensions. To avoid nonspecific staining, single cells Naïve and suture-inflamed BALB/c corneas were harvested and immersed in PBS containing 20 mM EDTA (Sigma-Aldrich) at 37°C for 30 minutes, and the corneal epithelium was removed with forceps to allow investigation of the corneal epithelium and stroma separately. Following two washes with PBS, the corneal stroma was cut into pieces and digested with 2 mg/mL collagenase D (Roche, Indianapolis, IN, USA) and 2 mg/mL DNAse I (Sigma-Aldrich, St. Louis, MO, USA) in DMEM for 60 minutes at 37°C. After digestion, corneal single cell suspensions were passed through a 70-μm cell strainer, and corneal epithelia and stroma were pooled into one and two separate samples per state, respectively, and blocked with Fc-block (as above).
Protocol tips
Samples were stained with fluorophore-conjugated antibodies against cell surface markers CXCR4. Single cell suspensions were then washed, fixed, and permeabilized (Cat. 555028; BD Bioscience)
Downstream tips
Single cell suspensions were then stained with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), and following a final wash underwent flow cytometric analysis using BD LSR II Cytometer (BD Biosciences). Acquired data were analyzed by FlowJo v10 (FlowJo, LLC, Ashland, OR, USA).

Publication protocol

"BALB/c spleens from naïve and tumor mice were mechanically digested and placed through a 70-μm cell strainer to obtain single cell suspensions. To avoid nonspecific staining, single cells were incubated and blocked with 1% Fc-block for 30 minutes at room temperature. Splenocytes were then labeled with fluorophore-conjugated antibodies against CD11c (clone N418, Cat. 117307; BioLegend), CXCR4 (clone 2B11, Cat. 53-9991-80; eBioscience), CD8 (clone 53-6.7, Cat. 100725; BioLegend), CD103 (clone 2-E7, Cat. 121409; BioLegend), and CD11b (clone M1/70, Cat. 101228; BioLegend) or appropriate isotype controls. Following a final wash, stained single cells were fixed with 4% paraformaldehyde (Cat. 15710; Electron Microscopy Sciences) and then analyzed using a BD LSR II Flow Cytometer (BD Biosciences).

Naïve and suture-inflamed BALB/c corneas were harvested and immersed in PBS containing 20 mM EDTA (Sigma-Aldrich) at 37°C for 30 minutes, and the corneal epithelium was removed with forceps to allow investigation of the corneal epithelium and stroma separately. Following two washes with PBS, the corneal stroma was cut into pieces and digested with 2 mg/mL collagenase D (Roche, Indianapolis, IN, USA) and 2 mg/mL DNAse I (Sigma-Aldrich, St. Louis, MO, USA) in DMEM for 60 minutes at 37°C. After digestion, corneal single cell suspensions were passed through a 70-μm cell strainer, and corneal epithelia and stroma were pooled into one and two separate samples per state, respectively, and blocked with Fc-block (as above). Samples were stained with fluorophore-conjugated antibodies against cell surface markers CXCR4 (2B11, Cat. 53-9991-80; eBioscience), CD45 (30-F11; BioLegend), CD11c (HL3; BD Biosciences), F4/80 (BM8; BioLegend), or appropriate conjugated isotype controls. Single cell suspensions were then washed, fixed, and permeabilized (Cat. 555028; BD Bioscience) and labeled with CXCL12 (Cat. 14-7992-83; eBioscience). Single cell suspensions were then stained with an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), and following a final wash underwent flow cytometric analysis using BD LSR II Cytometer (BD Biosciences). Acquired data were analyzed by FlowJo v10 (FlowJo, LLC, Ashland, OR, USA). Data are presented as relative expressions compared with isotype controls."

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