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BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation). When fluorochrome conjugated antibodies are bound to CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).
The ICRF44 monoclonal antibody specifically binds to CD11b, the 165-kDa adhesion glycoprotein that associates with the 95-kDa integrin β2 (CD18) to form the CD11b/CD18 complex, also known as Mac-1 or CR3. CD11b is a type I transmembrane glycoprotein that is encoded by ITGAM (Integrin alpha M). It is expressed on activated lymphocytes, monocytes, granulocytes, and a subset of NK cells. CD11b functions in cell-cell and cell-substrate interactions and is a receptor for iC3b, CD54 (ICAM-1), CD102 (ICAM-2) and CD50 (ICAM-3). This antibody significantly inhibits polymorphonuclear leukocyte aggregation in response to fMLP.
This clone also cross-reacts with granulocytes, a subset of peripheral blood lymphocytes and some monocytes of baboon, and both rhesus and cynomolgus macaque monkeys. The distribution on lymphocytes and granulocytes is similar to that observed with peripheral blood from normal human donors. There are fewer CD11b-positive monocytes present in the non-human primate blood than in normal human donor samples.
The antibody was conjugated to BD Horizon BV711 which is part of the BD Horizon Brilliant™ Violet family of dyes. This dye is a tandem fluorochrome of BD Horizon BV421 with an Ex Max of 405-nm and an acceptor dye with an Em Max at 711-nm. BD Horizon BV711 can be excited by the violet laser and detected in a filter used to detect Cy™5.5 / Alexa Fluor® 700-like dyes (eg, 712/20-nm filter). Due to the excitation and emission characteristics of the cceptor dye, there may be moderate spillover into the Alexa Fluor® 700 and PerCP-Cy5.5 detectors. However, the spillover can be corrected through compensation as with any other dye combination.
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