The IntelliCyt iQue Screener PLUS platform is a high-throughput FACS analyser which is fully integrated with robotics and high-throughput equipment which enables rapid, high content, multiplexed analysis of cells and beads in suspension in both 96 and 384 well plates. The instrument is a voltage-independent system, where altering voltages between plates is not required. Samples are delivered to detectors in an “air gap delimited stream”, and an entire plate of data is processed at one time. As little as 1 μL can be taken from assay wells without dead volume. Another feature of the instrument is the enhanced speed of acquisition – minimum sampling time for a 96 and 384 well plate is ~5 minutes and ~20 minutes, respectively. This makes it highly attractive option for researchers who want to analyse many samples quickly, as well as perform high throughput screens in suspension cells/bead assays. Data is acquired (and can be analysed) using the ForeCyt software, developed specifically to analyse plate-based data rapidly.

Enhancing the productivity of iQue® HTS, the 21 CFR Part 11 Compliance-Enabling Module (especially when handling clinical samples) supports the maintenance of electronic records with integrated global event tracking, electronic signatures, and additional compliance features.

Laser Information

The iQue instrument has 3 lasers, and 15 detection channels, with a dynamic range of > 7 decades. The laser/detection configurations are summarised in the table below:

Image

Preparing Your Sample Plate

Please note that the intelliCyt iQue PLUS Screener will only be operated by ACTD staff – no exceptions. Prior to the first run, clients are required to provide ACTD staff an empty plate for plate calibration. Clients are also required to provide unstained control, single color stained compensation controls and samples all in 96 or 384 well plate accompanied by a plate map which details what flourophores are present in each sample. This information should ideally be provided at the time of booking the instrument. 

V or U bottom vs flat bottom sample plate: the normal sampling position of the iQue instrument is from the centre of the well. When using flat bottom plates, however, the vortexing action of the shaker can cause the sample to accumulate on the edges of the well so it becomes necessary to sample from the side or corner of the well. The ACTD staff will configure sampling probe offset location for your desired plate.

Suggested volume per well (to be optimised based on project): 

• 20-30 µL/well in a 384 well plate
• 30-50 µL/well in a 96 well plate

Cell concentration: For cell based assays, we recommend a final cell concentration of 1 to 3×10⁶ cells/mL. Higher concentrations may clog the instrument.

Acquisition time: Please note that intelliCyt iQue PLUS Screener can only define acquisition time per well but not “stop event” per well for population of interest. Clients are recommended to make a calculation based on your total cell concentration and the percentage of population of interest (determined in advance) to decide acquisition time per well, but will ultimately need to be optimised for each specific project/condition. As a guide (in general):

• The collection of 1000-2000 events of interest per well is enough for data analysis/statistical relevance in most assays.
• When the peristaltic pump speed is set at 29 RPM* (standard acquisition speed), the instrument samples between 1.5 – 2 µL cell suspension per second.
• Calculate acquisitin time per well, for example:
  • Cell concentration = 1000 cells/μL (1x10⁶ cells/mL).
    Events of interest (e.g. T cells in mouse lymph nodes) = ~65% of total
    cells = 650 cells/ μL
    2 second acquisition (Sip) = ~3.5 μL = ~2,275 normal singlets/well.

*Note: the peristaltic pump speed (related to the acquisitin speed) can be optimised to obtain the best resolution for a specific project. 

Data analysis & export: we recommend analysis of data using the ForeCyte software package, which is specifically designed to enable the easy analysis and visualisation of plate data (e.g. IC50 curves, other graphs and hit identification can be utilised within the program). A copy of this software is available on our image analysis computer (to book, use the bookbrf.anu.edu.au website). Data can also be exported into FCS files and analysed using other common FACS based programs (e.g. FlowJo, FlowLogic). Exported data can also be shared to clients using CloudStor. Please speak to the ACTD team for further information.