Screening project assessing characteristics of suspension cells using flow cytometry

UiO is offering a suspension cell-based screening project using a flow cytometry based analysis with automated sample preparation. Potential readouts for the screen include basic cell characteristics like size and granularity, assessment of recombinant proteins (e.g. GFP), cell surface markers and intracellular staining like cell cycle analysis.

UiO has a strong background in immunology, cancer immunology and cell signaling analysis based on flow cytometry methods and is equipped with bio safety level 2 cell culture labs to handle primary human cells as well as established human cell lines. It has established a pipeline for the automated preparation of cell samples for flow cytometry in 384 format based on a Beckman Coulter Biomek i7 with an integrated plate centrifuge.

This setup is capable of running different protocols including preparation steps like cell fixation and permeabilization using various reagents like paraformaldehyde, saponine and methanol to prepare samples for extra and intracellular staining with agents including conjugated antibodies and DNA staining agents. Depending on volume requirements, staining reagents can be transferred using either the Biomek i7, liquid dispensers or an Echo acoustic liquid handler for volumes down to 2.5 nL. Samples will be analyzed on an Intellicyt (Sartorius) iQue screener PLUS flow cytometer equipped with 3 lasers (13 color channels).  Possible readouts for the flow cytometry screen include, but are not limited to basic cell characteristics like size and granularity, expression of recombinant proteins (e.g. labelled with GFP), cell surface markers (e.g. CD molecules) and DNA stain for cell cycle analysis.

Number of compounds screened

100,000 (full commercial set) of the EU-OPENSCREEN ERIC compound library

Time frame for screen up to validated hits

9 months

• Automated sample preparation using a Beckman Coulter Biomek i7 with 384 multichannel pipetting head plus span 8 and integrated Agilent V-spin plate centrifuge.
• Transfer of staining reagents using a BioTek EL406 washer/dispenser with BioStack 4 plate stacker (for volumes 1-2 µL/well).
• Transfer of staining reagents Labcyte Echo 550 acoustic liquid handler integrated into a Labcyte Access Workstation (for volumes 2.5 nL-1 µL).
• Data acquisition using an  Intellicyt  iQue screener PLUS high throughput flow cytometer, capable of running 384 well plates equipped with 3 lasers  (blue, red, violet) and 6 shared PMTs allowing 13 color channels plus FSC/SSC.
• Data analysis using ForeCyt flow cytometry software (Intellicyt, Sartorius) and KNIME.

• Validation of the assay provided by the user to ensure robustness (flow cytometry / iQue screener PLUS).
• Miniaturization of the assay into 384-well plates and automation of sample preparation (sample preparation / Biomek i7, flow cytometry/ iQue screener PLUS).
• Pilot screen using 5,000 compounds from the EU-OS compound library (sample preparation / Biomek i7, flow cytometry/ iQue screener PLUS).
• Analysis of pilot screen data (ForeCyt flow cytometry analysis software and KNIME).
• Full screen with the remaining 95,000 compounds of the EU-OS compound library (sample preparation / Biomek i7, flow cytometry/ iQue screener PLUS).
• Data analysis and subsequent selection of primary hits (ForeCytflow cytometry analysis software and KNIME).
• Hit picking of primary hits for hit validation (ForeCyt and KNIME).
• Validation of hits by determining IC50 (sample preparation / Biomek i7, flow cytometry / iQue screener PLUS).

Please note that this project includes the possibility of re-screening during potential chemical optimization under the medicinal chemistry call starting in 2021 (tentative date).

The prerequisite starting point to qualify for access is the availability of an established bioassay and associated key bespoke reagents, developed at lab-scale by the successful applicant. Criteria to define anestablished bioassay are compatibility with 96-well format (assay volume up to 150 µL), demonstrated successful flow cytometry based analysis by the applicant (e.g. successful detection of recombinant proteins by flow cytometry and not a different method like western blotting) and the availability of positive and negative controls.

Key bespoke reagents include the assay ready cells or particles that will be analyzed by flow cytometry (e.g. for expression analysis of recombinant proteins, the applicant has to provide the transfected cells, cell culture can be carried out on site) and special staining reagents like commercial kits and antibodies.

As specifics of the assay transfer procedure may vary between partner sites, the applicant and the individual sites will agree on the appropriate steps and logistics together.

Apply now

Topics / Keywords:

drug discovery, small molecules, high-throughput screening, cell-based assays

Open Time:

Start date:
June-01-2019 (20:00 CET)

Closing date:
September-30-2019 (20:00 CET)

Contacts:

Scientific / Technical / Methods contact:
johannes.landskron@ncmm.uio.no, alexandra.gade@ncmm.uio.no