Inverted epifluorescence microscope

Forces were measured on the stage of an inverted epifluorescence microscope (Leica) equipped with objectives of 63x (PL FLUOTAR; NA/0.7; C PLAN NA/0.75) and with a cooled CCD C5985 (Hamamatsu) or Coolpix 5000 camera (Nikon) as previously described [58 (link)]. To preserve intact the cell surface proteins, cells were dissociated with Cell dissociation enzyme-free buffer (Gibco, NY, USA) and then transferred in working medium (CO2-independent medium, Invitrogen, CA, USA) and used immediately. Two isolated cells were put into contact and allow forming adhesion for specific period times. Aspiration was monitored continuously by a pressure sensor (model DP103-38; Validyne) during the separation process and the values recorded for each of the last two cycles in the series (Pn-1 and Pn) were used to calculate the separation force (SF) for each doublet using the equation: SF = π (d/2)2 (Pn-1+Pn)/2 where d is the inside diameter of the left pipette. Results for 15–30 measurements were used to obtain the mean SF for a specific contact time.

Alkaline single cell electrophoresis assays for SS nicking was performed as described [88 (link)] using the CometAssay kit (Trevigen, Gaithersburg, MD). Briefly, A549 cells were transfected with siRNAs and treated with 10 mM HU for 1 h or mock treated. Cells were harvested, washed with cold PBS and mixed with molten 1:10 (v/v) LMAgarose and immediately spread over the sample area of comet slides. Cells were immobilized at 4°C in the dark for 30 min and immersed in lysis solution overnight. For the alkaline comet assay, slides were treated with alkaline unwinding solution for 1 h at 4°C in the dark before electrophoresis in alkaline electrophoresis buffer. Samples were rinsed with water and immersed in 70% ethanol before drying at 37°C for 15 min. SYBR Gold was used to stain dried agarose for 30 min at RT before rinsing and drying again. Slides were viewed with a Leica inverted epifluorescence microscope and analyzed by software Image J with OpenComet plugin [89 (link)]. Alkaline comet assays were performed five times in triplicate, counting >100 slides per experiment.

A micromanipulation technique was used to measure adhesion forces between cells as described previously [33 (link)]. Briefly, forces were measured on the stage of an inverted epifluorescence microscope (Leica) equipped with objectives of 63× (PL FLUOTAR; NA/0.7; C PLAN NA/0.75) and with a cooled CCD C5985 (Hamamatsu, Massy, France) or Coolpix 5000 camera (Nikon). Cells were manipulated at 37 °C with two micropipettes, each held by one micromanipulator connected to a combined hydraulic/pneumatic system and a pressure sensor making it possible to control and measure the aspiration applied to the cells. Micropipettes were pulled (model P-2000; Sutter Instrument, Novato, CA, USA), cut and fire-polished with a homemade microforge, such that their i.d. was 4.0–5.5 µm. The aspiration applied to the left pipette was measured using a pressure sensor (model DP103-38; Validyne, Northridge, CA, USA). Aspiration was monitored continuously during the separation process and the values recorded for each of the last two cycles in the series (P_n−1 and P_n) were used to calculate the separation force (SF) for each doublet using the equation: SF = π(d/2)² (P_n−1 × P_n)/2, where d is the i.d. of the left pipette. Results for around 20 measurements were used to obtain the mean force of separation for a specific contact time.