Purified human LDL (d=1.019-1.063), either native, acetylated, or oxidized, is reacted with a lipid stain, or a protein stain, for the production ofthe lipoprotein probes. The purified probes are ultrafiltered through a membrane, and packaged asceptically under nitrogen.The probes are provided in a solution with 154 mM NaCl and 0.34 uM EDTA, at pH 7.2. The protein concentration is 0.5 mg/mL. The purity of the probes is assessed by agarose gel electrophoresis.
DiI (1,1′-dioctadecyl- 3,3,3′,3′-tetramethylindocarbocyanine perchlorate) is a fluorescent dye which permeates the lipid portion of the lipoprotein particle. DiI is a carbocyanine dye that has been used extensively for labelling membranes. It is only slightly fluorescent in aqueous solutions, but highly fluorescent in a lipid environment. DiI has been excited at 488nm or 514nm and the emission is detected at 565 nm. These probes can be detected with a fluorescent microscope, fluorescent plate reader, or flow cytometer.
DiO (3,3′-dioctadecyloxacarbocyanine) is a lipophylic fluorescent dye which is excited at 484 nm and emits at 501 nm. These probes can be detected with a fluorescent microscope, fluorescent plate reader, or flow cytometer for multi-color fluorescent anaylsis.
Murine resident peritoneal macrophage and infiltrating macrophage were obtained by peritoneal lavage. Macrophage were incubated separately with 20 µg of each the probes for two hours. The cells were washed and analyzed by flow cytometry. The overlaid single parameter cytograms present the binding of DiI-Oxidized-LDL or DiI-Acetyl-LDL compared to the background binding of native LDL. Data provided courtesy of Dr. A. Palini, Responsabile, Servizio di Citometria, U.O. Immunoematologia e Medicina Transfusionale, H San Raffaele, Via Olgettina 60, Milano, Italia.
Biotin is a small molecule, covalently linked to the protein portion of the lipoprotein particle. A spacer group between the protein moiety of the lipoprotein and the biotin ensures its availability to aqueous solutions of avidin or streptavidin. Biotinylated materials are usually detected thru the secondary binding of an avidin or streptavidin conjugate.
Kalen now offers other lipoproteins with fluorescent probes.
Please contact Kalen for the availability of other fluorescent LDL probes as well as HDL probes. Send us an email at firstname.lastname@example.org