REAGENTS
NaCl (Sigma, Cat. N°. S3014)
KCl (Sigma, Cat. N°. P4504)
Na2HPO4 (Sigma, Cat. N°. S5136)
KH2PO4(Sigma , Cat. N°. P5655)
HCl (Sigma, Cat. N°. H1758)
HEPES (Sigma, Cat. N°. H4034)
Ultraglutamine 1, 200 mM in 0,85% NaCl solution (Lonza, Cat. N°. BE17-605E/U1)
Antibiotic-antimycotic 100x (GIBCO, Invitrogen, Cat. N°. 15240)
Ampicillin sodium salt (SIGMA- ALDRICH, Cat. N°. A9518-56)
Kanamycin B sulfate salt (Sigma, Cat. N°. B5264-250M6)
Trypsin, 0.25% (1X) with EDTA (Invitrogen, Cat. N°. 25200-072)
Fetal Bovine Serum E.C. Approved (GIBCO, Invitrogen, Cat. N°. 10106)
Acridine Orange (Calbiochem, MERK, Cat. N°. 113000-1GM)
Mounting medium for microscope preparation EUKITT (Aname, Cat. N°. RT15320)
Inmersion liquid, type F (Leica microsystem, GmbH, Cat. N°. 11513859
Cell culture
Corning 75 cm2 Flask, Canted Neck (BD Biosciences, Cat. N°. H108CO2836)
Cell Culture Plate, 6-well, (BD Falcon™, Cat. N°. 353046)
Cell Culture Coverslip, Sterile, Thermanox plastic, 13 mm diameter (NUNC™ Brand Products, Cat. N°. 174950)
Microscope Slides, 76 x 26 mm (Menzel-Gläser, Cat. N°. AA00000112E)
Microscope Cover Slips, 24 x 36 mm (Menzel-Gläser, Cat. N°. BB024036A1)
Counting Chamber Improved Neubauer (BRAND, Cat. N°. 717805)
Sterile Pipette 10 ml (APL, Cat. N°. PN10E1), 1 ml (APL, Cat. N°. PN1E1)
Micropipette PIPETMAN P1000 (Gilson, Cat. N°. F123602), P100 (Gilson, Cat. N°. F123615), P10 (Gilson, Cat. N°. F144802)
Syringe Filter, Acrodisc (PAAL corporation , Cat. N°. PN 4433)
Syringe 5ml, luer slip (BD Discardit , Cat. N°. 309050)
Universal yellow tip 5-200 μl, blue tip 100-1000 μl, tip 1-5 ml (Daslab, Cat. N°. 162001, 162222, 162005) and tip 0,1-10 μl (Deltalab, Cat. N°. 200024)
Microtubes MCT-150-C 1,5 ml (Axygen Quality, Cat. N°. 311-08-051 )
Conical tube 15 ml (17 x 120 mm) (BD Falcon, Cat. N°. 352096)
Conical tube 50 ml (30 x 115 mm) (BD Falcon, Cat. N°. 352070)
Pasteur Pipettes - soda glass (230mm) (Deltalab, Cat. N°. 702)
Manual Counter cash. 4 digits (Quirumed, Cat. N°. 052-63047001)
Tweezers, style 62A (ANAME, Cat. N°. 78350-62A)
REAGENTS SETUP
Ampicillin stock 500x: Dissolve 500 mg of ampicillin powder in milli-Q H2O to a final volume of 100 ml. Filter-sterilized and store 1 ml aliquots at - 20ºC.
Kanamicyn stock 500x: Dissolve 500 mg of kanamicyn powder in milli-Q H2O to a final volume of 100 ml. Filter-sterilized and store 1 ml aliquots at -20ºC
HEPES stock 100x: Dissolve 26.029 g of HEPES and adjust to a final volume of 500 ml with milli-Q H2O and filter sterilize. Store at 4°C.
Acridine Orange solution: Dissolve 20 mg of acridine orange in 20 ml of PBS and then filter it and keep in the dark to avoid light. Before using, dilute it until 40 μg/ml in PBS. In order to obtain final concentration 10 μl of this solution is added to cultured medium.
Phosphate saline buffer (PBS): Dissolve 0.2 g of KCl , 0.2 g of KH2PO4, 8 g of NaCl , 1.15 g of Na2HPO4 in 1 L of milli-Q H2O and filter sterilize.
Cell lines
Mouse embryonic fibroblast cell line (NIH3T3) (ATCC, CRL-1658)
Normal human prostate epithelium immortalized with SV40 cells (PNT1A) (Sigma 95012614)
Chinese hamster ovary cell line (CHO) (ATCC, CCL-61)
Androgen-sensitive human prostate adenocarcinoma cell line (LNCaP) (Sigma-Aldrich, 89110211)
Human prostate carcinoma cell line (DU 145) (ATCC , HTB-81)
Androgen-insensitive human adenocarcinoma cell line (PC3) (ATCC, CRL-1435)
Human breast adenocarcinoma cells (MCF7) (ATCC, HTB-22)
Human epithelial cervical carcinoma cell line (HeLa) (Sigma Aldrich, 93021013)
Murine melanoma cell line (B16-F10) (ATCC, CRL-6475)
Mouse leukemic monocyte macrophage cell line (RAW 264.7) (ATCC , TIB-71)
Rat glioma cell line (C6) (ATCC, CCL-107).
Growth media and supplements
RPMI 1640 (Lonza, Cat. N°.BE12-167F sterile filtered) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM Ultraglutamine, 0,1% Ampicillin, 0,1% kanamycin and 15 mM HEPES.
DMEM (Dulbecco´s Modified Eagle´s Medium with 1 g/L Glucose, without L-Glutamine)(Lonza, Cat. N°.BER-707F) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM Ultraglutamine, 15 mM HEPES and 1% GIBCO antibiotic-antimycotic cocktail.
DMEM/F12 (Dulbecco´s Modified Eagle´s Medium, Ham´s F-12 1:1 mix with 15 mM Hepes and L-Glutamine) (Lonza, Cat. N°.BE12-719F) supplemented with 10% Fetal Bovine Serum (FBS) and 1% GIBCO antibiotic-antimycotic cocktail.