We stained single cell suspensions following standard procedures using fluorochrome-conjugated antibodies supplied by eBiosciences and directed against CD3 (145-2C11), TCRA/B (H57-597), CD4 (RM4-5), CD8a (53-6.7), PD1 (J43), ICOS (C398.4A), CD25 (7D4), CD69 (H1.2F3), CD86 (B7-2) (GL1), Ly-6G (Gr-1) (RB6-8C5), CD11b (M1/70), CD19 (1D3), ICOSL (HK5.3), BCL6 (K112-91) and FOXP3 (150D/E4). The antibody against CD45R (B220) (RA3-6B2) is from Miltenyi and the anti-IFNG (XMG1.2) was purchased from BD Biosciences. For detection of CXCR5, we used purified anti-CXCR5 antibody (2G8) from BD Biosciences and followed a three-step staining protocol as previously described (Johnston et al., 2009 (link)). Intracellular detection of BCL6 and FOXP3 were performed under standard conditions using the FOXP3 transcription factor staining buffer (eBiosciences) as directed by the manufacturer’s protocol. For flow cytometry detection of phosphorylated intracellular proteins, cells were fixed with 4% formaldehyde and permeabilized with 90% ice cold methanol and then incubated with phosphor-ERK (197G2), phosphor-AKT (D9EXP) and phosphor-S6 rabbit antibodies (D68F8) followed by secondary antibody staining using an anti-rabbit Alexa-647 antibody, all supplied by Cell Signaling. We acquired flow cytometry data using a FACS Canto cytometer (BD Biosciences) and analyzed them using FlowJo software (TreeStar).
Alexa 647 antibody
Manufactured by Cell Signaling Technology
The Alexa-647 antibody is a fluorescently labeled secondary antibody used in various immunodetection techniques. The antibody is conjugated to the Alexa Fluor 647 dye, which emits in the far-red/near-infrared range of the spectrum, making it suitable for applications such as flow cytometry, immunofluorescence, and Western blotting.
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Lab products found in correlation
Fluorochrome conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Anti ifng xmg1.2, by BD (1 mentions)
Phosphor erk 197g2, by Cell Signaling Technology (1 mentions)
Facscanto cytometer, by BD (1 mentions)
Fix perm solution, by Thermo Fisher Scientific (1 mentions)
Phospho histone h3 ser10 antibody, by Cell Signaling Technology (1 mentions)
Software, by FlowJo (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Facscanto 2, by FlowJo (1 mentions)
2 protocols using alexa 647 antibody
1
Multiparameter Flow Cytometry Immune Profiling
2
Quantifying Apoptosis and Cell Proliferation
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For annexin-V and Phospho-Histone H3 (pSer12 (link)) detection, the forelimb bud of embryos at time 0 or cultured for 1, 3 and 6 hours were harvested and incubated for 15 minutes in trypsin, 5% EDTA (Invitrogen). Then, tissues were disrupted mechanically and the released cells washed in complete media. For detection of annexin-V detection, cells were washed with binding buffer and incubated with annexin-V FITC (BD Pharmingen, Le Pont-de-Claix, France) during 15 minutes at room temperature. For Phospho-Histone H3 detection, cells were permeabilized with the intranuclear detection Perm/Fix solution (eBioscience) and stained 1 hour with the Phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology). Then, cells were washed and stained 30 minutes with anti-rabbit Alexa 647 antibody (Cell Signaling Technology). Finally, samples were acquired on the FACS Canto II and analyzed using the FlowJo software (Ashland, OR, USA).
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