Combining fluorescent cell barcoding and flow cytometry-based phospho-ERK1/2 detection at short time scales in adherent cells.

Detection of levels of intracellular phospho-proteins is key to analyzing the dynamics of signal transduction in cellular systems. Cell-to-cell variability in the form of differences in protein level in each cell affects signaling and is implicated in prognosis of many diseases. Quantitative analysis of such variability necessitate measuring the protein levels at single-cell resolution. Single-cell intracellular protein abundance detection in statistically significant number of adherent cells for short time sampling points post stimulation using classical flow cytometry (FCM) technique has thus far been a challenge due to the detrimental effects of cell detachment methods on the cellular machinery. We systematically show that cell suspension obtained by noninvasive temperature-sensitive detachment of adherent cells is amenable to high-throughput phospho-ERK1/2 protein detection at single-cell level using FCM in these short time sampling points. We demonstrate this on three adherent cell lines, viz., HeLa, A549, and MCF7, from distinct lineages having characteristically different elasticity at 37 °C. In particular, we use a right combination of multiplexing via fluorescent cell barcoding (FCB) and intracellular antibody staining for simultaneous detection of phospho-ERK1/2 (pERK) stimulated by epidermal growth factor (EGF) in multiple samples. Based on systematic characterization using Alexa 350 dye, we arrive at two conditions that must be satisfied for correct implementation of FCB. Our study reveals that the temperature-sensitive detachment of HeLa cells correctly captures the expected pronounced bimodal pERK distribution as an early response to EGF, which the enzymatic treatment methods fail to detect.