MDA PCa 2b and NCh660 cells had been cultured Hams F12 medum wth 20% FBS, 25 ng ml choleratoxn, ten ng ml EGF, five mM phosphoethanolamne, 0.1 ng mlhydrocortsone, 45 nM selenc acd and five mg ml nsulne.All cells had been propagated at 37uC conventional cell culture condtons.dentty of cell lnes was confrmed by arrayCGH oAgent 244humagenome arrays, just after ten?15 passages cells had been dscontnued.Mnaturzed 3D cultures.Cells have been embedded betweetwo layers of Matrgel ouncoated Angogeness m sldes bottom wells were fled wth 10 ml of Matrgel culture medum and polymerzed at 37uC for thirty mn.Cells have been theseeded at 20.000 cells ml densty.After attachment, cells were covered wth a second layer of Matrgel culture medum, allowed to polymerze overnght at 37uC.Cell culture medum was altered every 2nd day.3D bulk cultures for RNA extracton.Prostaspheres were cultured Mlcellhangng cell culture nserts wth one.0 mm PET transparent membranes o6 very well plates.Membranes have been pre coated wth Matrgel medum and ncubated at 37uC for 1h, to prevent attachment towards the membrane.
Cell suspensowas mxed 14 wth Matrgel, transferred towards the coated well, and polymerzed overnght at 37uC.Cells had been fed just about every other day wth fresh medum from beneath.Cell fxaton, mmunofluorescence labelng and magng.Mnaturzed 3D cultures have been fxed wthmcrowells,usng 4% paraformaldehyde, supplemented wth 0.8% TrtoX one hundred, 5 mM EGTA and 1 mM MgCl2 for 15?twenty mnutes at RT.Fxed cultures were washed 3 tmes wth PBS and blocked for 1h wth 20%horse serum.Cultures had been ncubated overnght at 4uC wth prmary selleck antbodes, washed wth PBS, and ncubated at area temperature for 4h wth secondary antbodes andhoechst nuclear stan.3D structures were staned wth CalceAM lve cell dye.Confocal three dmensonal mages were takeby usng Zess Axovert 200 M wth spnnng dsc confocal untokogawa CSU22 and also a Zess PlaNeofluar 56 objectve.Z stacks had been acqured wth a stesze of 19 mm.ntensty projectons have been designed by SldeBook 4.two.0.7 and NH mageJ, even further analyzed wth VTT Acca software.
Box plots had been vsualzed wth R.20x phase contrast tme lapse mages were acqured wth ncucyte, pre processed wth chloroxine mageJ and analyzed wth VTT Acca.RNA extractoand mcroarrays.3D bulk cultures were washed wth ce cold PBS, membranes excsed wth a scalpel, and spherods transferred nto 6 effectively plates.Gels had been mxed vgorously wth 9 ml of five mM EDTA PBS, transferred nto 15 ml Falcotubes, and ncubated oa tabletorocker for 45 mto detach in the Matrgel.Prostaspheres have been sedmented by centrfugatoand lysed wth RLT buffer.Cells propagated monolayer had been lysed at 90% confluence,

drectly from ten cm cell culture dshes usng RLT buffer.Total RNA was extracted wth RNeasy Mn kt, accordng for the suppliers protocol.300 ng RNA was amplfed wth Ambons lumna TotalPrep RNA Amplfcatokt.VT reactowas carried out overnght toeld suffcent botnylated cRNA.