Phospholipidosis detection
From LipidomicsWiki
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= Phospholipidosis detection in human macrophages using NBD-PE = | = Phospholipidosis detection in human macrophages using NBD-PE = | ||
| - | Human monocytes were isolated from healthy, normolipidemic volunteers with the apolipoprotein E3/E3 genotype by leukapheresis followed by counterflow elutriation. Cells were cultured at 37°C/5% CO2 at 70.000 cells/well in 96 well plates (Becton Dickinson) with 200μl/well macrophage serum-free medium (Invitrogen, Karlsruhe, Germany) containing recombinant human monocyte-colony stimulating factor (M-CSF) 50 ng/ml (R&D Systems, Minneapolis, Minnesota) to induce phagocytic differentiation. Four day M-CSF differentiated human monocyte derived macrophages were cultured 24h with E-LDL (40 | + | Human monocytes were isolated from healthy, normolipidemic volunteers with the apolipoprotein E3/E3 genotype by leukapheresis followed by counterflow elutriation. Cells were cultured at 37°C/5% CO2 at 70.000 cells/well in 96 well plates (Becton Dickinson) with 200μl/well macrophage serum-free medium (Invitrogen, Karlsruhe, Germany) containing recombinant human monocyte-colony stimulating factor (M-CSF) 50 ng/ml (R&D Systems, Minneapolis, Minnesota) to induce phagocytic differentiation. Four day M-CSF differentiated human monocyte derived macrophages were cultured 24h with E-LDL (40 µg/ml) or Ox-LDL (60 µg/ml) and 12µg/ml of the fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) (Molecu-lar Probes, Eugene, OR, USA). Amiodarone (15μmol/L) and amitryptilin (30μmol/L) were used as a positive control. The final concentration of DMSO vehicle was 0.3% which does not affect NBD-PE accumulation of cytotoxicity results. The method was adapted from (1). Afterwards cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times with PBS.<br>The method was adapted from Morelli JK et al Cell Biol Toxicol 22 (2006) 15-27.<br> |
[[Image:Phospholipidosis.png]] | [[Image:Phospholipidosis.png]] | ||
Current revision
Phospholipidosis detection in human macrophages using NBD-PE
Human monocytes were isolated from healthy, normolipidemic volunteers with the apolipoprotein E3/E3 genotype by leukapheresis followed by counterflow elutriation. Cells were cultured at 37°C/5% CO2 at 70.000 cells/well in 96 well plates (Becton Dickinson) with 200μl/well macrophage serum-free medium (Invitrogen, Karlsruhe, Germany) containing recombinant human monocyte-colony stimulating factor (M-CSF) 50 ng/ml (R&D Systems, Minneapolis, Minnesota) to induce phagocytic differentiation. Four day M-CSF differentiated human monocyte derived macrophages were cultured 24h with E-LDL (40 µg/ml) or Ox-LDL (60 µg/ml) and 12µg/ml of the fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) (Molecu-lar Probes, Eugene, OR, USA). Amiodarone (15μmol/L) and amitryptilin (30μmol/L) were used as a positive control. The final concentration of DMSO vehicle was 0.3% which does not affect NBD-PE accumulation of cytotoxicity results. The method was adapted from (1). Afterwards cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed three times with PBS.
The method was adapted from Morelli JK et al Cell Biol Toxicol 22 (2006) 15-27.
Figure 3: Phospholipidosis detection using NBD-PE
Images show phospholipidosis detection with NBD-PE in M-CSF differentiated, E-LDL and Ox-LDL loaded and with amitryptilin teated macrophages as a positive control for phospholipidosis induction.
