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Human DiI-Labeled Very Low Density Lipoprotein


Cat.#:  10463

   Size:   2.0 mg

One Week Lead Time

          Product Description          
Background:   Purified LDL is labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, and then isolated by ultracentrifugation. The resultant product is exhaustively dialyzed against phosphate buffered saline, (pH 7.4), sterilized by membrane filtration and then aseptically packaged in a solution containing phosphate-buffered saline at pH 7.4 and 0.2 mM EDTA. Each lot is evaluated on a murine macrophage cell line for fluorescence uptake.
Storage & Stability: DiI-VLDL is stable for 6 weeks after receipt when handled aseptically and stored at 2-8°C (Don’t Freeze). Note: After prolonged storage, some precipitate may be observed. This is normal for the product. Spin in centrifugation at 5000×g for 10 minutes before using.
Tested Applications: DiI-VLDL are evaluated for receptor binding to peritoneal macrophages in conjunction with VLDL and Ox-VLDL.
  Typical Lipoprotein Labeling Protocol  
  1. Aseptically dilute the DiI-VLDL to 10-50 μg /ml in your culture media.
  2. Add to live cells and incubate for 2-5 hours at 37°C.
  3. Remove media containing DiI-VLDL from your culture.
  4. Wash cells several times with probe-free media.
  1. Fluorescence Microscopy:
  2. Visualize using standard rhodamine excitation: emission filters (or suggested wavelengths excitation:emission at 554nm:571nm or near). 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.
  3. Cell Sorting:
  4. 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: 554 nm Emission: 571 nm

Fixation and Mounting of DiI Labeled Cells  
  1. Wash 3 times in PBS.
  2. Fix in 3% formaldehyde/PBS for 20 minutes at room temperature.
  3. Rinse 5 seconds in distilled water at room temperature.
  4. Drain liquid onto chem-wipe.
  5. Invert cover, slip on a drop of 90% Glycerol and 10% PBS onto a microscope slide.
  6. Seal with Kroenigs wax, also known as cover glass cement (Pfaltz & Bauer, Waterbury, CT 06708). Do not use nail polish. Store at -20°C.
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