Single Cell Analysis

MCF7 (ATCC) cells were cultured using high glucose Dulbecco’s Modified Eagles Medium (DMEM; ThermoFisher Scientific, UK) supplemented with 10% (v/v) foetal bovine serum (FBS; ThermoFisher Scientific, UK) in polystyrene flasks in a 5% CO₂ 37 °C cell incubator. Suspensions of single cells are prepared by detaching cells in culture using Accutase solution (Sigma, UK).

A microfluidic based method was used to facilitate single cell protein analysis based on previously reported work⁴. The PDMS microfluidic chip is fabricated using well known methods of soft-lithography⁴³. It is formed of a main channel through which single cell suspensions are flowed to which an array of analysis chambers (n = 50) are connected. Each analysis chamber has dimensions 300 μm × 300 μm × 35 μm resulting in an assay volume of 3.15 nL. The chip is sealed using a functionalised coverslip (Nexterion; Schott, Europe) upon which an anti-p53 antibody (Enzo Life Sciences, UK) microarray is printed using an OmniGrid Micro microarrayer (Digilab, UK). The microarray spotting solution contained the anti-p53 capture antibody mixed 1:1 with a print buffer formed of 3 × saline-sodium citrate buffer, 1.5 M betaine supplemented with 0.01% SDS; the final concentration of antibody in the spotting solution was 0.5 mg mL⁻¹. The spots are printed at defined locations which allowed them to be aligned to the PDMS chip such that a single antibody spot is aligned to the centre of each analysis chamber. The chip is filled with a solution of 0.25 μg mL⁻¹ fluorescent detection antibody (anti-p53 DO1 labelled with Alexa Fluor 488; Santa Cruz, USA) with 4% bovine serum albumin in phosphate buffered saline.

The platform has been described in detail elsewhere⁴. An inverted microscope (Nikon Ti-E; Nikon, Japan) forms the basis of the experimental platform. Single cells are individually isolated into analysis chambers using an optical trap (1070 nm YLM-5 Ytterbium fibre laser; IPG Photonics, UK). Once all analysis chambers are occupied, single cell lysis is achieved optically by launching a high energy laser pulse (6 ns pulse 1064 nm Surelite SLI-10 Nd:YAG; Continuum, USA) into the medium immediately surrounding the cell. The pulse produces an expanding cavitation bubble which shears the cell membrane allowing intracellular contents to be released. Antibody spots are imaged using objective-based total internal reflection fluorescence (TIRF) microscopy (488 nm Versalase; Laser 2000, UK) and an electron-multiplied CCD camera (IXON DU-897E; Andor Technologies, Ireland). The acquired images are analysed using the methods above.