Dil ac ldl

Briefly, mice were euthanized with an overdose of CO₂ before cells were isolated from Rptor^EC−/−, Rictor^EC−/− or Mtor^EC−/− mice, and their wild-type littermates using positive immuno-selection with a rat anti-mouse CD31 (BD). EC purity was examined with the uptake of Dil-Ac-LDL (Biomedical Technologies) and FITC-conjugated anti-CD31 (Invitrogen) labeling. Primary purified aortic EC were cultured in EGM2 (Lonza)^{50 (link)}.

Cells were incubated for 4 hours with acetylated LDL labeled with 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL; Biomedical Technologies) at 37°C in order to evaluate cellular functionality for LDL uptake. The test was performed according to the manufacturer's instructions after which cells were visualized using a Leica DMRA microscope (Leica).

PBMNCs were labeled with 20 µg/ml of acetylated low-density lipoprotein and 1,10-dioctadecyl-3,3,30,30-tetramethyl-indocarbocyanine perchlorate (Dil-Ac-LDL) (Biomedical Technologies, Inc., Stoughton, MA, USA) at a concentration of 4 × 10⁴ cells/500 µl for 30 min in a 37 °C CO₂ incubator. The PBMNCs were centrifuged down at 400 g for 10 min followed by washing with 2% FBS/PBS and suspension in 2% FBS/PBS at a concentration of 1 × 10³ cells/50 µl. The labelled PBMNCs were then cocultured with human umbilical vein endothelial cells (HUVECs) at an MNC-to-HUVEC ratio of 1 × 10³-to-1.5 × 10⁴ cells in a final volume of 100 µl. The cell mixture was incubated in a 37 °C water bath, and then 100 µl of the cell mixture was transferred into a pre-coated Matrigel (thin coat method) 50 µl/well in 96-well plates and incubated at 37 °C in a CO₂ incubator for 10 h. The assessment of tube formation was performed using a Nikon Ti-S Intensilight Ri1 NIS-D inverted fluorescence microscope (Nikon Instruments). All experiments were performed in triplicate. The intensity of fluorescence from incorporated labeled PBMNCs from QQ culture media and from standard culture media in HUVECs was compared [11 (link)].

The morphology of EPCs in different growth periods was observed. Adherent cells were characterized by the uptake of Dil-acLDL (Biomedical Technologies, Stoughton, MA, USA) and FITC-labeled UEA-1 lectin (Vector Labs, Burlingame, CA, USA) staining. Cells were also characterized by co-immunofluorescence staining for CD31 and KDR expression. After staining, the samples were viewed under an inverted fluorescence microscope (Leica, Germany). The ability of passage 3–5 cells (late EPCs) to form capillary-like tubes in vitro was assessed on Matrigel.

Second-generation DB/+-EPCs and DB/DB-EPCs were incubated at room temperature with 10 μg/ml Dil-Ac-LDL (Biomedical Technologies, Stoughton, MA) for 4 h and then fixed in 4% paraformaldehyde for 15 min. The fixed cells were washed twice in PBS (pH 7.4). Subsequently, cells were incubated for 1 h with 10 μg/ml FITC-UEA-1 (Molecular Probes, USA). The images of fluorescently labeled cells were captured using a laser scanning confocal microscope (Leica, Germany). Cells positive for both markers are considered EPCs [30 (link)].