- Enter the average absorbance value from each experimental
duplicate into the rearranged equation (as they-value), and
then proceed to solve equation to produce the known protein
concentration. - Normalize disintegrations per minute (DPM) to milligram of
protein for each sample (seeNote 14).
3.3 acLDL Uptake 1. After BMDM have differentiated, seed cells at a density of
2.5 105 cells per 24-well.
- The following day, remove medium and wash cells with
1 PBS. - Add 0.5 mL of BMDM medium containing 10 μg/mL
Dil-acLDL, and incubate in a humidified 5% CO 2 incubator
at 37C for 1–2 h. - Remove the medium and wash twice with 1PBS.
- Add 250μL of isopropyl alcohol to each well, seal plates with
parafilm, and cover with aluminum foil. - Incubate plate at room temperature for 10 min and then at
4 C overnight with gentle rocking. - Collect isopropyl alcohol from each well in a 1.5 mL microfuge
tube (seeNote 15). - Load 70μL on a fluorescence compatible 96-well plate.
- Add a serial dilution of Dil-acLDL as standards to calibrate
fluorescence, as well as non-Dil-acLDL loaded samples and
isopropyl alcohol controls. - Read the plate using a fluorometric plate reader with an excita-
tion of 545 nm and emission wavelength of 580 nm (or com-
parable fluorescent plate reader) (seeNote 16).
3.4 Cholesterol
Efflux
- After BMDM have differentiated, seed cells at 2.5 105 cells/
well in 24-well plates. - Incubate lipid-loading DMEM with 1μCi/mL^3 H-cholesterol
at 37C overnight to conjugate. - The following day, remove the media from the cells and wash
with 1PBS. - Add 0.5 mL of the lipid-loading medium supplemented with
1 μCi/mL^3 H-cholesterol to each well (seeNote 17). - Incubate cells in a humidified 5% CO 2 incubator at 37C for
30 h to induce acLDL uptake and accumulation. - Remove the medium and wash the cells twice with 1PBS to
remove all exogenous lipid and radioactivity. - To allow time for the cholesterol pools to equilibrate, add
0.5 mL of equilibration medium.
Assessing Macrophage Cholesterol Homeostasis 487