Diaphot

Senescence was assessed by SA-β-gal staining of HCT116 cells transfected with indicated mimics or expression vectors. After 24hrs post transfection the cells were cultured in complete and serum free media followed by staining using senescence β-galactosidase staining Kit (Cell Signaling, Danvers, MA) following manufacturer's instructions. Briefly, the cells were rinsed with PBS and fixed (2% formaldehyde, 0.2% glutaraldehyde in PBS) for 15 min at room temperature and washed twice with PBS. The cells were incubated with fresh β-galactosidase staining solution at pH 5.8 and incubated at 37°C for overnight. SA-β-galactosidase positive cells were detected by inverted bright field microscopy (Nikon Diaphot) at 100x magnification.

The Hcn2 and SkM1 currents (I_Hcn2 and I_SkM1, respectively) were measured in the whole-cell configuration of the patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices Corporation, Sunnyvale, CA, United States). CPCs were harvested, stored in SP++ medium at room temperature (20°C), and studied within 4 h. Cell suspensions were put into a recording chamber on the stage of an inverted microscope (Nikon Diaphot), and single CPCs that visibly formed branches with the bottom of the recording chamber and exhibited green fluorescence were selected for electrophysiological measurements. Voltage control, data acquisition, and analysis were accomplished using custom software. Potentials were corrected for the estimated change in liquid-junction potential (Barry and Lynch, 1991 (link)). Signals were low-pass filtered with a cut-off frequency of 5 kHz and digitized at 5 and 20 kHz for I_Hcn2 and I_SkM1, respectively. Series resistance was compensated by ≥80%. Cell membrane capacitance (C_m) was calculated by dividing the time constant of the decay of the capacitive transient after a −5 mV voltage step from −40 mV by the series resistance, and amounted to 24.8 ± 2.5 pF (n = 18).

Glass shards containing plated cells were placed in a recording chamber mounted on an inverted microscope (Nikon Diaphot, USA). The cells were continuously superfused with a pre-warmed (37 °C) standard Tyrode's solution containing (in mM) the following: 140 NaCl, 4 KCl, 2.5 CaCl₂, 1 MgCl₂, 10 Glucose, 5 HEPES (titrated to pH 7.4 with NaOH). A modified “High K⁺” version of this solution (containing 94 mM KCl and 50 mM NaCl) was used to elicit recordable currents from T623A, G648A and F656A hERG channels mutants [34,37] . Glass patch-pipettes (Schott #8250 glass, A-M Systems Inc., USA) were pulled (Narishige, PP 830) and polished (Narishige, MF 83) to obtain a final resistance between 2 and 4 MΩ. Patch-pipettes were dialysed with an intracellular solution containing (in mM) the following: 130 KCl, 1 MgCl₂, 5 EGTA, 5 MgATP, 10 HEPES (titrated to pH 7.2 with KOH). All recordings were made using an Axopatch 200B amplifier (Axon Instruments, now Molecular Devices) and a CV203BU head-stage. Pipette resistance compensation was between 70 and 80%. Data were acquired using a Digidata 1320 interface (Axon Instruments, now Molecular Devices). Data digitization rates were 10–25 kHz during all voltage protocols and an appropriate bandwidth of 2–10 kHz was set on the amplifier.

Embryos were morphologically evaluated on days three and five of development
using an inverted Nikon Diaphot microscope with a Hoffmann modulation contrast
system (Eclipse TE 300 microscope, Nikon, Tokyo, Japan) under 400x
magnification.
The blastocyst development rate was defined as the number of embryos that reached
blastocyst stage at day five by the number of fertilized oocytes in each cycle.
On day 5, one to two embryos were transferred per patient, depending on maternal
age and embryo quality, using a soft catheter with transabdominal ultrasound
guidance.