Protocol for optimizing surface expression and speed in coaggregation assays using K562 cells and subsequent analysis with a CoAg Index

Abstract

Coaggregation assays using K562 cells have been extensively employed to study how cell adhesion molecules mediate specificity between different populations. Here we describe how to prepare K562 cells, optimize electroporation conditions, calibrate antibodies used for protein detection, determine the surface expression of desired adhesion molecules, and considerations for the rotational force to be applied during the assay. We also detail procedures for analyzing coaggregates using our established CoAggregation (CoAg) Index. For complete details on the use and execution of this protocol, please refer to Bisogni et al.¹.

Keywords: cell biology; cell culture; cell-based assays.

Graphical abstract

Figure 1

Electroporated K562 cells imaged with the Zoe Fluorescent Imager (A and B) Healthy low passage K562 cells (A) electroporated with an eGFP-N1 construct (B). (C and D) Unhealthy higher passage K562 cells (C) electroporated with an eGFP-N1 construct (D) can produce false aggregates. Scale bar = 100 μm.

Figure 2

Typical expected outcomes (A) Confocal image obtained with a Zeiss LSM510 of a coaggregation assay involving two populations of K562 cells transfected with different combinations delta protocadherins fused to GFP or tagRFP. Images have been pseudocolored yellow and pink for ease of visualization. (B) Example surface biotinylation optimization assay to demonstrate balanced surface expression of plasmids co-transfected into K562 cells. Scale bar = 100 μm.

Figure 3

Flow chart demonstrating the steps of the CoAg Index macro Images have been pseudocolored for ease of visualization. (A) Starting individual image. (B) Binarized image of (A). (C) Denoised image of (B). (D) Partitioning of (C). Scale bar = 100 μm.