DiI-LABELED?ACETYLATED?LOW?DENSITY?LIPOPROTEIN,?HUMAN

Catalog?No: SA007

Lot?No: 2015-04-20

Quantity: 500ug(micrograms)Protein/Vial

Concentration: 3.0mg/ml?(Protein)

Introduction?

DiI-Ac-LDL,?Acetylated?Low?Density?Lipoprotein,?labeled?with?1,1-dioctadecyl?–?3,3,3,3-tetramethyl-indocarbocyanine?perchlorate,?labels?both?vascular?endothelial?cells?and?macrophages.??It?can?be?used?to?identify?and/or?isolate?these?cells?from?mixed?cell?populations.??When?cells?are?labeled?with?DiI-Ac-LDL,?the?lipoprotein?is?degraded?by?lysosomal?enzymes?and?the?DiI?(fluorescent?probe)?accumulates?in?the?intracellular?membranes.??Labeling?cells?with?DiI-Ac-LDL?has?no?effect?on?cell?viability.?Pure?cultures?of?vascular?endothelial?cells?can?be?isolated?from?complex?primary?cultures?using?fluorescent?activated?cell?sorting?based?on?their?increased?metabolism?of?the?DiI-Ac-LDL.?Contaminating?cell?types?(fibroblasts,?smooth?muscle,?pericytes,?epithelial?cells)?are?not?labeled.??Macrophages?can?be?differentiated?from?mixed?cell?populations?(including?endothelial?cells)?because?they?are?more?brightly?labeled.?

Labeling?endothelial?cells?with?DiI-Ac-LDL?has?many?advantages?over?labeling?other?endothelial?cell?associated?antigens.??The?labeling?procedure?is?one?step,?and?once?the?cells?are?labeled,?the?fluorescent?probe?(DiI)?is?not?removed?by?Trypsin.?Both?low?density?and?confluent?cultures?of?vascular?endothelial?cells?are?effectively?labeled.?No?other?cell?type?(other?than?macrophages)?is?labeled?to?the?same?level?as?vascular?endothelial?cells.??Each?lot?of?DiI-Ac-LDL?is?evaluated?for?the?specific?labeling?of?bovine?aortic?endothelial?cells?and?murine?macrophages?to?assure?consistent?results.??A?complete?labeling?protocol?is?included?with?each?shipment.?We?also?offer?an?"FITC-like"?label?DiO-Ac-LDL,?which?is?useful?for?fixed?wavelength?FACS?Cell?sorters.?

Storage?&Stability:

This?product?is?stable?for?6?weeks?when?handled?aseptically?and?stored?at?2-8°C.??

PROTECT?FROM?LIGHT?AND?NEVER?FREEZE.

*Special?Note: After?prolonged?storage,?some?precipitate?may?be?observed.??This?is?normal?for?this?product.?Clarify?out?the?aggregates?by?spinning?in?a?microfuge?for?2?minutes.

*Preparations?of?DiI-Ac-LDL?are?fairly?unstable;?plan?your?experiments?in?advance?and?use?fresh?material.

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Procedural?Outline?

1.?Dilute?DiI-Ac-LDL?to?20-50ug/ml?in?growth?media.

2.?Add?to?cells?and?incubate?for?4?hours?at?37oC.

3.?Remove?media.?

4.?Wash?with?probe-free?media.?

5.?Visualize?via?Fluorescence?Microscopy?and/or?trypsinize?(or?EDTA)?for?cell?sorting.?

6.?Fluorescence?Microscopy:

Visualize?using?standard?rhodamine?excitation:?emission?filters?(or?suggested?wavelengths?excitation:emission?at?549nm:565nm).?If?fixation?is?desired?use?3%?formaldehyde?in?PBS.??(Never?use?methanol?or?acetone?fixation?-?DiI?is?soluble?in?organic??solvents).

Note:??A?positive?culture?must?be?stained?for?comparison?purposes.

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B.?Cell?Sorting:

Label?as?in?steps?1-5.?Trypsinize?or?treat?cultures?with?EDTA?to?produce?a?single?cell?suspension.

Use?labeled?pure?cultures?of??positive?and?negative?cell?types?to?set?gates?on?the?cell?sorter.

Suggested?Wavelengths?for?Cell?Sorting:???Excitation:??488/514/549nm；Emission:???565nm