Counting Proteins in Single Cells with Addressable Droplet Microarrays

Summary

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Abstract

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Introduction

The goal of this method is to determine the variation in abundance of a target protein in a cell population with single cell resolution. Single cell analysis provides a number of benefits that are not available with traditional ensemble biochemical methods.^1,2,3,4,5 Firstly, working at the single cell level can capture the rich heterogeneity of a cell population that would otherwise be lost by the averaging that occurs with traditional ensemble biochemical techniques. The majority of work-horse biochemical ....

Protocol

1. Preparation

1. Make chips and print antibody microarrays
   1. Attach an adhesive silicone/acrylic isolator to a coverslip functionalized to support an antibody microarray. This is referred to as the chip.
      NOTE: Various surface chemistries have been tested for their suitability with addressable droplets.⁵ Surface chemistries may need to be optimized for alternative capture agents. ADM isolators are available commercially or may be produced by laser cutting acrylic (CAD file for isolator used in this work is provided as a download; see the Supplemental Code File).
   2. Switch on t....

Results

The absolute basal protein copy number of p53 was determined with single cell resolution in a human colon cancer cell line, BE cells. We demonstrate how p53 expression can vary over several orders of magnitude and show a weakly positive correlation between cell size and protein copy number within the resting BE cell population.

Addressable Droplet Microarrays are formed when aqueous droplets are dispensed at antibody spot locati.......

Discussion

Addressable Droplet Microarrays are a sensitive and extensible method for quantitatively determining the absolute copy number of protein within a single cell.

Limiting the level of non-specific binding (NSB) is critical within the protocol to achieving as low a limit of detection as possible. Proteins and other biochemical species may non-specifically bind to a number of interfaces present within the droplets — the coverslip surface, the antibody spot and the oil/water interface. Protein.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture
Phosphate-Buffered Saline (PBS)	Life Technologies	10010015
DMEM high glucose	Sigma	D6429
Foetal Bovine Serum (FBS)	Biochrom	S0115
cell culture flasks	Corning	SIAL0639
Trypsin/EDTA	Biochrom	L2153
Name	Company	Catalog Number	Comments
Microarray
Microcontact Arrayer	DigiLab, UK	OmniGrid Micro
Microcontact pin	ArrayIt, USA	946MP2
Coverslips (Nexterion)	Schott, Europe	1098523	Size (mm): 65.0 x 25.0; Thickness (mm) 0.17
p53 capture antibody	Enzo	ADI-960-070
p53 detection antibody, Alexa Fluor 488 labelled	Santa Cruz	sc-126	stock concentration 200μg/mL
Saline-sodium citrate buffer	Gibco	15557-044
Betaine	Sigma	61962
Sodium dodecyl sulphate	Sigma	L3771
384 well plate (low volume)	Sigma	CLS4511
Nitrogen gas cylinder	BOC	Industrial grade, oxygen-free
Name	Company	Catalog Number	Comments
Droplets
Micromanipulator	Eppendorf	Patchman NP2
Manual Microinjector	Eppendorf	CellTram Vario
Micropipette	Origio, Denmark	MBB-FP-L-0
Syringe pumps	KD Scientific	KDS-210
100 μL syringe	Hamilton	81020	Gas tight, PTFE Luer lock
1 mL syringe	Hamilton	81327	Gas tight, PTFE Luer lock
Silicone isolator	Grace Bio-Labs	JTR24R-A-0.5	6x4 well silicone isolator with adhesive
Laser cutter	VersaLASE	VLS2.30 CO2 Laser 3W	for laser cutting of custom isolators
1mm thick acrylic sheet	Weatherall-UK	Clarex Precision Sheet 001	for laser cutting of custom isolators
Adhesive sheet	3M		used to adhere custom isolators to microarrayed coverslips
Super glue	Loctite	LOCPFG3T
150 μm ID/360 μm OD fused silica tubing	IDEX	FS-115
1.0 mm ID/1/16” OD PFA tubing	IDEX	1503
0.014” ID/0.062” OD PTFE tubing	Kinesis	008T16-100
1.0 mm ID/2.0 mm OD FEP tubing	IDEX	1673
Bovine Serum Albumen (BSA)	Fisher Scientific	BP9700100
Mineral oil	Sigma	M5904
Ultra-pure water	Millipore, Germany	MilliQ
Name	Company	Catalog Number	Comments
Microscopy & Optics
TIRF microscope with encoded XY stage	Nikon, Japan	Nikon Ti-E
EM-CCD	Andor Technologies, Ireland	IXON DU-897E
Laser excitation source	Vortran, USA	Stradus 488-50
Optical lysis laser source	Continuum, USA	Surelite SLI-10
Microscope filter cube for TIRF	Chroma, USA	z488bp
Microscope filter cube for Optical Lysis	Laser 2000, UK	LPD01-532R-25
Name	Company	Catalog Number	Comments
Software
Fiji	Open Source		Image analysis software
Matlab	Mathworks	version 7.14 or higher	Image analysis software