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Tissue/ cell preparation (Human). (Peripheral blood mononuclear cells).
Our Abpromise guarantee covers the use of ab8220 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 0.1µg for 106 cells.
The antibody is excellent marker of activated myeloid cells.
ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|IP||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration. Use under non-reducing conditions.|
|Blocking||Use at an assay dependent concentration. PubMed: 23039186|
|Functional Studies||Use at an assay dependent concentration. Induces homotypic cell aggregation and high affinity LFA1 conformation in resting leukocytes.|
Human peripheral blood lymphocytes stained with ab8220 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab8220, 0.1μg/1x106 cells) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.
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