DiI-HDL DiI标记高密度脂蛋白

Dil-High Density Lipoprotein DiI标记高密度脂蛋白

High Density Lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate.

Cat No:C02640

Size: 500μg/vial

Concentration: Based on actual label concentration.Buffer containing phosphate-buffered saline at pH 7.4 and 0.01 mM EDTA.

Specifications: 0.22 micron membrane filtered,aseptically filled.Cell Culture Tested.

Absorbance Ratio: DiI/Protein=484nm/275nm=1.21

Storage:

This product is stable for 6 weeks after receipt when handled aseptically and stored at 2-8℃(Do not free).HDL products have a natural tendency to aggregate.Aggregates of this product can interfere with its use.To clarify these aggregates out,simply spin in centrifuge tube for 2 minutes.

Product Preparation:

Purified HDL is labeled with the fluorescent probe,Dil,and reisolated by ultracentrifugation(1.063- 1.21g/mL).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 μM EDTA.Each lot is evaluated on a murine macrophage cell line for fluorescence uptake.

Typical Lipoprotein Labeling Protocol

1.Aseptically dilute the Dil-HDL to 20-40 μg/mL in growth media.

2.Add to live cells and incubate for 4-5 hours at 37℃.

3.Remove media containing Dil-HDL from your culture.

4.Wash cells several times with probe-free media.

A.Fluorescence Microscopy:

Visualize using standard rhodamine excitation:emission filters (or suggested wavelengths excitation:emission at 484nm:501nm).If fixation is desired use 3%formaldehyde in PBS.(Do not use methanol or acetone fixation -Dil is soluble in organic solvents).

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

B.Cell Sorting:

Label as 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:484nm.Emission:501nm.

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.Do not use nail polish.Store at -20℃.

Special note:

1. Do not freeze.
2. LDL products have a natural tendency to aggregate.Aggregates of this product can interfere with its use.To clarify these aggregates out,simply spin in a microfuge for 2 minutes.
3. For research use only.