High throughput screening: A beginner’s guide to new age science wizardry

Science can often be tedious and time consuming, eating up hours upon hours of pipetting and processing. With the advancement of technology, we are now able to automate these processes which allows for the potential of high throughput screening. This technique takes advantage of robotics to rapidly process thousands of samples with incredible consistency and precision. Not only do these factors save time, but they eliminate the possibility of human error between samples, increasing the robustness of results. One of the main problems with high throughput screening is that many scientists think of it as a technique used only by Big Pharma, or very large research centers.

This blog post aims to provide a point of view walk through of a screen (through the eyes of someone who has also never run one!) searching for a small molecule TNF-α inhibitor: a much sought after therapeutic for chronic inflammatory diseases. Here in we will walk through and exhibit the relative simplicity of the actual protocols of running this screen while hopefully demonstrating its usability to a wider scientific audeience.

The Technique
The most important step in a screen is to properly plan your experiment, as High throughput screens are very expensive. Our experiment aims to administer the Sigma LOPAC drug library to HeLa cells to access if any of these molecules can inhibit TNF-α induced NFκB translocation to the nucleus.

-HeLa human cervical carcinoma cells were seeded at 5200 cells/well in 10x 384 well plates (5 sets in duplicate) in 18µL DMEM. Cells were incubated with either:

LOPAC¹²⁸⁰ small molecule inhibitor library (Sigma) which are all characterized to be biologically active
Bortezomib (Santa Cruz #sc-217785) – 26S proteasome inhibitor which acts as a negative control by                                                                                      blocking Ikk degradation and therefore NFκB translocation.
DMSO – drug loading control to verify DMSO suspending drugs isn’t toxic to our cells
Water – to make sure TNF is working
*All liquid manipulations performed by a robotic Janus Workstation (Perkin Elmer)


* Figure generated by Dr. Edan Foley. University of Alberta. Department of Medical Microbiology and Immunology. MMI 590 lab manual. Page 29. Figure 3.

-Cells were then incubated at 37°C for 30 minutes.
-Recombinant TNFα (Sigma T0157) was then added to each well at 10ng/mL and further incubated for 1 hour
-Medium was aspirated and cells were washed with PBS.
-Cells fixed in 3.7% Formaldehyde for 20min (room temperature)
-Formaldehyde aspirated
-Cells then incubated with 50uL (dispensed with plate washer) PTX buffer (1xPBS, 0.01% Triton X-100) and incubated for 5 minutes (x3 with new PTX).
-15µL of blocking buffer (1x PBS, 5% Normal goat serum, 0.1% Tween-20) was added (using the workstation)
-Cells incubated for 30 minutes then blocking buffer was dumped from plates
-Added 15µL of 1:1000 Rabbit anti-NFκB (Santa Cruz D5796)
-Incubated overnight at 4°C
-Dumped primary antibody solution and washed x4 with PBT (1xPBS, Tween-20) using the plate washer
-added 15µLof 1:1000 Hoechst 33258 (Molecular probes H-3569) 1:1000 Goat anti-rabbit AF488 (Life Tech A11008) and incubated 1h at room temperature in the dark
-Antibody solution was dumped and plates were washed with PBT twice and once with PBS before being stored in PBS for subsequent analysis on a Perkin Elmer Operetta high content imaging system

More info on the sample analysis and subsequent discussion to come!