Gurr s buffer

DMEM/F12 (1X), phosphate-buffered solution (PBS) 1X without calcium or magnesium, Corning glutagro supplement (200 mM), tissue culture dishes, flasks and plasticware were purchased from Corning (Corning, NY). Sodium pyruvate (100 mM) was purchased from Lonza (Allendale, NJ). Gurr’s buffer and 0.5% trypsin-EDTA (10X) were purchased from Life Technologies Corp (Carlsbad, CA). Crystal violet and acetic acid were purchased from J.T. Baker (Phillipsburg, NJ). Lead chromate (CAS #7758-97-6), sodium chromate (CAS#7775-11-3), and demecolcine were purchased from Sigma-Aldrich (St. Louis, MO). Giesma stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Seradigm premium grade fetal bovine serum (FBS), Sodium dodecyl sulfate (SDS), and methanol were purchased from VWR International (Radnor, PA). Potassium chloride (KCl) was purchased from Alfa Aesar (Tewksbury, MA). Attachment factor was purchased from ThermoFisher Scientific (Waltham, MA). Trace-element grade nitric acid was purchased from Fischer Scientific (Hampton, NH).

DMEM/F12 (1X), phosphate-buffered solution (PBS) 1X without calcium or
magnesium, glutagro supplement (200 mM), tissue culture dishes, flasks and
plasticware were purchased from Corning (Corning, NY). Cosmic Calf Serum (CCS)
was purchased from Hyclone (Logan, Utah). Sodium pyruvate (100 mM) was purchased
from Lonza (Allendale, NJ). Gurr's buffer and 0.5% trypsin-EDTA (10X) and
0.25% trypsin-EDTA (1X) were purchased from Life Technologies Corp (Grand
Island, New York). Zinc chromate (CAS# 13530-65-9, 99.7% purity) was purchased
from Pfaltz and Bauer (Z00277, Waterbury, Connecticut). Crystal violet and
acetic acid were purchased from J.T. Baker (Phillipsburg, NJ). Demecolcine was
purchased from Sigma-Aldrich (St. Louis, MO). Giesma stain was purchased from
Biomedical Specialties Inc. (Santa Monica, CA). Seradigm premium grade fetal
bovine serum (FBS), Sodium dodecyl sulfate (SDS), and methanol were purchased
from VWR International (Radnor, PA). Potassium chloride (KCl) was purchased from
Alfa Aesar (Tewksbury, MA). Attachment factor was purchased from ThermoFisher
Scientific (Waltham, MA). Trace-element grade nitric acid was purchased from
Fischer Scientific (Hampton, NH).

Sodium chromate, lead chromate, colcemid, and potassium chloride were purchased from Sigma-Aldrich (St. Louis, MO). Gurr’s buffer, trypsin/EDTA, sodium pyruvate, penicillin/streptomycin, and L-Glutamine were purchased from Invitrogen (Carlsbad, CA). Giemsa stain was purchased from Biomedical Specialties (Santa Monica, CA). Sodium dodecyl sulfate (SDS) was purchased from American Bioanalytical (Natick, MA). Crystal violet, methanol and acetone were purchased from J.T. Baker (Phillipsburg, NJ). Dulbecco’s minimal essential medium and Ham’s F-12 (DMEM/F-12) were purchased from Mediatech (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Tissue culture dishes, flasks, and plasticware were purchased from Corning (Acton, MA)

Sodium chromate, Triton X-100, demecolcine and potassium chloride (KCl) were purchased from Sigma Chemical (St. Louis, MO). Giemsa stain was purchased from Ricca Chemical Company. (Arlington, TX). Crystal violet, sodium dodecyl sulfate (SDS), and acetic acid were purchased from J.T. Baker (Phillipsburg, NJ). Nitric acid and methanol were purchased from BDH Chemicals (Radnor, PA). Gurr’s buffer, Keratinocyte-SFM, and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Corporation (Grand Island, NY). Tissue culture dishes, flasks and plasticware were purchased from BD (Franklin Lakes, NJ). Urothelial cell medium, trypsin/EDTA and trypsin neutralizing solution were purchased from ScienCell Research Laboratories (Carlsbad, CA). Penicillin/streptomycin was purchased from Mediatech (Manassas, VA). Paraformaldehyde 4% was purchased from Alfa Aesar (Ward Hill, MA). Phospho-histone H2A.X (Ser139) antibody was purchased from Cell Signaling (Beverly, MA). AlexaFluor 488-conjugated goat anti-rabbit IgG secondary antibody was purchased from ThermoScientific (Rockford, IL).

RPMI was purchased from Mediatech (Manassas, VA). Penicillin/streptomycin, Gurr’s buffer, and trypsin/EDTA were purchased from Invitrogen Corporation (Grand Island, NY). Crystal violet, acetic acid, and methanol were purchased from J.T. Baker (Phillipsburg, NJ) and Fetal Bovine Serum (FBS) was purchased from Gibco Life Technologies (Grand Island, NY). Tissue culture dishes, flasks, and plasticware were purchased from BD (Franklin Lakes, NJ). Lead chromate, sodium chromate, potassium chloride (KCl) and demecolchicine were purchased from Sigma/Aldrich. Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA).

RPMI was purchased from Mediatech (Manassas, VA). Penicillin/streptomycin, Gurr’s buffer, sodium, L-glutamine and trypsin/EDTA were purchased from Invitrogen Corporation (Grand Island, NY). Dulbecco’s minimal essential medium and Ham’s F-12 (DMEM/F12) 50:50 mixture was purchased from Mediatech Inc. (Herndon, VA). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Fetal Bovine Serum (FBS) was purchased from Gibco Life Technologies (Grand Island, NY).Crystal violet, acetic acid, and methanol were purchased from J.T. Baker (Phillipsburg, NJ). Tissue culture dishes, flasks, and plasticware were purchased from BD (Franklin Lakes, NJ). Lead chromate (PbCrO₄), sodium chromate (NaCrO₄), potassium chloride (KCl), and demecolchicine were purchased from Sigma/Aldrich (St. Louis, MO) Giemsa stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Cytoseal 60 slide mounting medium was purchased from VWR (Bridgeport, NJ). MycoAlert detection kits were purchased from Lonza Rockland, Inc (Rockland, ME).

Three iPSC clones were chosen for karyotyping. IPSC lines were plated in a 6-well plate. Each well was treated with 20 μL colcemid (GIBCO) for 2 hours in a 37°C incubator to arrest the mitotic cells in metaphase. Colonies were lifted with Accutase (Stem Cell Technologies, Vancouver, British Columbia, Canada) and subsequently centrifuged at 850 rpm for 3 minutes. The cell pellet was resuspended in 0.067 M KCl (Sigma) hypotonic solution and incubated for 20 minutes at room temperature. A 3:1 methanol (Fisher Scientific):acetic acid (Sigma-Aldrich) fixative solution was added to the hypotonic solution and incubated for 5 minutes at room temperature. This was followed by 3 treatments in a 3:1 methanol (Fisher Scientific):acetic acid (Sigma-Aldrich) fixative solution, with each treatment incubated for 1 hour at room temperature. Samples were dropped onto clean wet slides and were aged in an oven at 90°F for 1–2 hours. Afterward, slides were immersed in Trypsin (HyClone) for 30–40 seconds and rinsed in FBS and saline. After an additional rinse in saline, slides were stained in 12.5% Giemsa (GIBCO) in Gurrs buffer (GIBCO) for 2–3 minutes. Slides were rinsed in distilled water and air-dried. Microscopic analysis included scanning of all slides, the count of a minimum of 20 metaphases, analysis of a minimum of 7 metaphases, and karyotyping a minimum of 2 metaphases.