Candida albicans-induzierte Genexpression in primären ... - OPUS

2.10 siRNAs (Doppelstrang-Oligonukleotide) .....................................................23
2.11 Zellen, Pilze und Bakterien .........................................................................24
2.12 Geräte.........................................................................................................25
3 Methoden ..........................................................................................................27
3.1 Zellkultur .....................................................................................................27
3.1.1 Lagerung, Einfrieren und Rekultivierung von Zellen.............................27
3.1.2 Zellkultur ..............................................................................................27
3.1.3 Herstellung verschiedener Präparationen von Candida albicans.........29
3.1.4 Stimulation und Inhibitorinkubation ......................................................29
3.2 Gentransfer in eukaryotische Zellen ...........................................................30
3.2.1 Retrovirale Infektion von HUVEC.........................................................31
3.2.2 DEAE-Dextran-Transfektion.................................................................31
3.2.3 Kalziumphosphat-Transfektion.............................................................32
3.2.4 Oligofectamine-Transfektion ................................................................32
3.2.5 Weitere Transfektionsmethoden ..........................................................33
3.3 Oligonukleotid-Mikroarray-Analyse .............................................................33
3.4 Quantitative real time RT-PCR....................................................................36
3.5 Lyse von Zellen zur Proteinanalyse ............................................................37
3.6 Western Blot ...............................................................................................38
3.6.1 SDS-Polyacrylamidgelelektrophorese (SDS-PAGE) ............................38
3.6.2 Proteintransfer......................................................................................39
3.6.3 Immundetektion....................................................................................39
3.6.4 Stripping...............................................................................................39
3.7 Enzyme-linked Immunosorbent Assay (ELISA) ..........................................40
3.8 Protein-Aktivierungsassay (Immunkomplex-Kinaseassay) .........................40
3.9 Promoter-Reporter-Assay (Luciferase-Assay) ............................................41
3.10 Durchflußzytometrie....................................................................................42
3.11 Molekularbiologische Methoden..................................................................43
3.11.1 Transformation von Plasmid-DNA in Bakterien....................................43
3.11.2 Mini-, Midi- und Maxipräparation von Plasmid-DNA.............................43
3.11.3 Sequenzierung von DNA......................................................................43
II