Karyomax colcemid solution

Cells were first arrested in metaphase with KaryoMAX Colcemid solution (Gibco) and then harvested after a treatment with a hypotonic salt solution. Two sets of probes were used to localize plasmid integration site. RP23-336p21 and RP24-129K5 specific bacterial artificial chromosomes (BACs) that map to the E3 band of the mouse chromosome 7 (Human BAC Clone Library, Children’s Hospital Oakland Research Institute [CHORI]) were used as controls. PGK-hFANCA transgene integration site was detected using as probe the DNA from the plasmid vector. Following the manufacturer’s specifications, BACs DNA was directly labelled by nick translation (Vysis, Abbott Molecular) with SpectrumGreen-dUTPs, whereas plasmid DNA was labelled with SpectrumOrange-dUTP (Vysis). The probes were blocked with Cot-1 DNA and DNA sheared salmon sperm (Vysis) to suppress repetitive sequences, and hybridized overnight as 37 °C onto metaphase spreads. After post-hybridization washes, the chromosomes were counterstained with DAPI in Vectashield mounting medium (Vector Laboratories). Cells images were captured using a cooled charge-coupled device (CCD) camera (Photometrics SenSys camera) connected to a computer running a Chromofluor image analysis system (CytoVision, Leica Biosystems).

For the cell cycle analysis, Muse™ Cell Cycle Kit (Merck, Kenilworth, NJ, USA) was used. Flp‐In™ T‐REx™ 293 cell lines expressing the corresponding transgene were grown on four 10‐cm plates per cell line until 50% confluency, at which point KaryoMAX Colcemid solution (Gibco, Dublin, Ireland) was added 1:100 to two plates per cell line for 6 h. Cells were harvested by trypsinization and fixed by resuspending in ice‐cold 70% EtOH. The final EtOH suspension was calculated to contain 2 million cells/ml. The resuspensions were kept at −20°C overnight. For cell counting, 300 μl of cell suspension was centrifuged at 450 g for 5 min and washed once with 1× PBS. Three hundred microliters of cell cycle reagent was added, and the solution was incubated at room temperature for 30 min in the dark. A total of 10,000 events were measured for each sample on Guava easyCyte single sample flow cytometer (Merck). The G2‐phase peak was defined using the KaryoMAX‐treated samples, while the results were obtained from untreated samples.

PC-iPS (P28) and PEF-iPS (P18) cells were passaged and cultured for 2 days, then the culture medium was replaced with 2 ml of fresh LCDMV medium with 200 μl KaryoMAX Colcemid Solution (15210-040; Gibco) in one 6-well, and cells were incubated at 37 °C under 5% CO₂ for 2.5 h. The PC-iPS and PEF-iPS cells were then collected and resuspended in 5 ml 0.075 M KCl (SCRC). After incubation for 30 min at 37 °C, 0.5 ml of fixative (methanol (SCRC) and acetic acid (SCRC) at 3:1 (v/v) ratio) was added to the KCl solution and centrifuged at 1000 rpm for 10 min at 4 °C. The supernatant was then removed, and 10 ml of ice-cold fixative was added. The cells were incubated on ice for 30 min, and the fixation step was then repeated but with on-ice incubation for 1 h. The final cell precipitation comprising a single-cell suspension was dropped on to microscope slides, incubated, treated with 0.01% Trypsin-EDTA, stained with the Rapid Giemsa Staining kit (E6073141; BBI Life Science), and photographed (Leica; DFC365 FX).

Epstein Barr Virus-transformed peripheral-blood lymphocytes (EBV-PBLs) of the two affected siblings, the healthy brother (no carrier), a healthy control line, as well as two established Fanconi cell lines (F1 and F2) were used to study chromosomal breakage. Fanconi patient F1 carries a homozygote pathogenic class 5 variant in FANCG (NM_004629.1), namely c.637_643delTACCGCC (p.Tyr213Lysfs*6). Fanconi patient F2 is combined heterozygote for two mutations in FANCC (NM_000136.2) namely c.520C > T (p.Arg174*) and c.455dupA (p.Asn152Lysfs*9), both class 5 mutations. Peripheral blood lymphocytes were cultured according to standard procedures. After 24 h of incubation, 20 nM Etoposide (Sigma E1383) was added to the culture for 27 h. After 24 h, cells were arrested in metaphase through a 3 h treatment with 10 μg/ml KaryoMAX Colcemid solution (Gibco), treated with 0.075 M KCl, fixed in methanol:acetic acid (3:1), spread onto glass slides and air-dried. Slides were stained with bisBenzimide H 33258 (Sigma), exposed to UV light and finally stained with Giemsa. For DEB analysis, the procedure is similar. Instead of Etoposide, a 0.004% diepoxybutane solution was added to the cultures after 24 h of incubation. In both cases, a minimum of 25 cells were analyzed and the number of breakages per mitosis was calculated.

To pause cell cycle at metaphase, the KaryoMAX^® Colcemid™ Solution (Gibco, Dublin, Ireland) was added to the culture medium at 1:100 dilution and the cells incubated at 37 °C for 24 h. The cells were then trypsinized and centrifuged. After discarding the supernatant, the pellet was re-suspended in 0.06 M KCl/citrate-saline solution and incubated at 37 °C for 20 minutes. Cells were fixed in methanol/acetic acid (3:1) twice. The cell suspension was placed on a glass slide and stained by DAPI (DOJINDO, Kumamoto, Japan), and the number of chromosomes were counted in the fluorescent images.

The media and reagents used to culture BM were Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Corporation, Carlsbad, CA, USA), fetal bovine serum (FBS; JRS Scientific Inc., Woodland, CA, USA), penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Australia), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-6 (IL-6) (Miltenyi Biotec, Bergisch Gladbach, Germany). The reagent, 1,4-BQ (CAS No: 106-51-4 and ≥ 98% purity) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Methylcellulose culture media were purchased from StemCell Technologies (Vancouver, BC, Canada) for culturing each progenitor lineages. The reagents used throughout the conventional karyotyping experiments were KaryoMAX Colcemid Solution, Trypsin EDTA 1× (Gibco, Grand Island, NY, USA), and Leishman stain (Sigma-Aldrich Co., St. Louis, MO, USA).