HCS Basics

If you are new to high content screening (HCS) and analysis (HCA) you may be overwhelmed with questions.  If your first question is “What is HCS?” then we encourage you to go to the Wikipedia definition of High-content Screening to find out more, but as they put simply.

High content screening is an automated cell biology method drawing on opticschemistrybiology and image analysis to permit rapid, highly parallel biological research and drug discovery.

There are so many moving pieces to a successful high content screen that it usually takes a team of people with a diverse array of specialized expertise to get that perfect set of data.   Follow links on key words below to learn more about these specialized areas or techniques as we walk through an example of some of the considerations that come into play during a typical high content screen.

Assay Development

Before you can do a HCS you need to have a robust assay.  This includes the following things.

  • Knowing what and why you want to measure a particular readout
  • Reproducible cell plating, reagent delivery, staining, and imaging protocols
  • Positive and negative controls for every plate and batch
  • An automated quantitative readout that is statistically significant
  • A reagent library for screening

Automation and Robotics

Unless you are doing a very small assay that you plan to execute with hand pipetting, you are likely going to be using automation to help you conserve reagent usage and costs, reduce plating artifacts, and tighten CVs.  Automation can be applied to each step of a HCS assay.

  • Cell plating and incubation
  • Reagent additions (compound, siRNA etc.)
  • Fixation and staining
  • Plate imaging and loading onto the microscope
  • Barcode tracking


Where would HCS be without the automated microscope?  Walking away from an imaging session of 30, 384 well plates takes careful set up of your imaging system and robotic plate handler.  Nothing ruins a screen faster than artifact laden or out of focus images.  Things to consider in the imaging room include

  • Autofocus routines and exposure times
  • Light intensity and evenness of illumination
  • Optimal multi-band pass filters and potential bleed through
  • Plate placement and removal on the microscope
  • Image naming and storage
  • Photobleaching and fluorophore lifetime during reimaging or large batches

Image Analysis

Quantitative cell biology requires data from many cells to be statistically significant.  While some people will spend hours scoring images, your best bet is to work with software or data analysis professionals that have tools for image segmentation, data capture, and storage.  Working out how to best quantify your target or label of choice is priority number one prior to running a screen and requires having images that best reflect the ones you will acquire during a screen with example control and reagent responses.  Some standard techniques include

  • DNA and whole cell segmentation
  • Quantifying fluorescent intensity, distribution, or shape
  • Trained recognition of a particular response pattern
  • Cell population numbers or migration
  • Kinetic changes

Data Handling

The HCS includes the collection of a vast array of disparate data.  LIMS and database solutions are your best bet for keeping track of all of the moving parts and putting them back together again when it’s time to analyze your screen.  Some things you will need to keep track of include

  • Cell plating and plate barcodes
  • Reagent concentrations, incubation times, well positions and barcodes (compounds, siRNA etc)
  • Image names, labels, and individual and/or average data for every image in every well
  • Plate and batch level control data
  • Replicate matching
  • Individual runs details – e.g protocols, operators, and programs used

Your Screening Library

Whether you are doing a compound, natural product, antibody, or siRNA screen you are going to need to work out strategies for getting samples from the main stock of the reagent library to your cells.  It is worth thinking ahead to the feasibility of your final screen during assay development so you don’t optimize something that is impossible in practice.  Considerations include:

  • Limitations of fluid handling aspiration and dispensing volumes
  • Conservation, contamination and dilution of stock reagent library
  • Concentration or dose response of addition
  • Plate maps and controls
  • Replicates

Hit Identification and Statistics

Knowing when a hit is a hit should be built into to every screen.  Weak signal to noise in your staining, image quality, drug response, off-target effects, concentration, plating CVs and otherwise can cast a doubt on the best data.  Some techniques commonly applied to sort through the data include

  • Z-score, t-test, redundant siRNA activity (RSA), RNAi gene enrichment ranking (RIGER), cSSMD
  • % relative to control
  • Within plate or between plate averages and changes

Cherry Picking and Confirmation Assays

Be sure to plan for going back to your library to confirm your hits and to work through how to get more reagent.  There is nothing more frustrating than a beautiful result that you can’t confirm.  Your library manager or vendor should be able to work through details on how to best acquire samples.  Confirmation assays may include

  • Dose response curves
  • Time course of response
  • Competition assays
  • Different cell lines
  • Other markers

Team High Content

So who is on your ideal team to execute a High Content Screen?

  • Cell and molecular biologists
  • Automation specialists
  • Reagent library managers (compounds, siRNA etc.)
  • Microscopists
  • Image analysis programmers
  • Statisticians
  • Database administrators and developers

The good news is that many vendors are out there who can provide users with tools and services that can often be used in a lab lacking one or more of these key players.  Keep your nose to the pages of High Content Review to learn how!