ImageStreamX MKII Imaging Flow Cytometer
The Amnis Imagestream X MKll is a powerful new technology added to our core which combines the principles of multicolor flow cytometry with the ability to capture the cells’ actual image while in the sheath fluid core, enabling a number of new ways of studying a sample. With this new technology one can look at nuclear translocation, co-localization, autophagy, and cellular spot counting. Unlike microscope based methods, one can see these cellular events within specific phenotypes using the principles of polychromatic flow cytometric analysis, or other flow cytometric gating strategy, while counting hundreds, or thousands of cells at rates of one to two thousand cells per second. Rare events can be very effectively studied and the data possible resulting from powerful image analysis algorithms using the “IDEAS” software suite, can be quite compelling.
The ImagestreamX MKll at Feinstein has four lasers; 488 nm, 405 nm, 561 nm, and 642 nm and is capable of collecting nine colors in addition to two Bright Field channels , and one Side Scatter channel for a total of twelve channels of data. The images generated are analyzed in a software program called “Ideas” which is available on an independent workstation within the facility, at no charge.
Please visit the Imagestream website to see a more in-depth description of the instrument, available support for sample preparation protocols, and data analysis techniques. The “Ideas” software is a powerful image analysis Windows only program available for download at NO CHARGE from the Amnis Imagestream website. The CPU requirements to successfully analyze images are extensive, and not commonly available on most desktops such that use of our own special CPU “Ideas” workstation is recommended and is available to users at no charge.
IDEAS software Dell Computer Workstation
IDEAS software suite performs high content morphometric analysis on tens of thousands of images collected on the ImagestreamX MKll.
- Cell boundaries are automatically determined
- Identify area of interest on the cell of interest – then apply to all cells
- Obtain individual cell data as well as population statistics
- Identify subtle shifts in staining patterns
- “Features” are what IDEAS uses to quantify cell morphology.
- 82 features per image
- 13 function masks
IDEAS software is available for free download through the Amnis Imagestream Customer Portal – be aware that your host CPU must satisfy some minimum requirements as the data analysis is quite intense and requires a good deal of computing power.
Web training for this software, as well as the software itself, is available as well as protocol development and applications support at the Amnis Imagestream Website.
Becton Dickenson FACSAria IIU and FACSAria Sorp
Running DIVA acquisition and analysis software. This instrument is a high speed cell sorter with capacity to separate cells into subpopulations sorting in up to four directions, or sorting for single cell deposition into a variety of multiwell plates. The sorter is equipped with UV, Violet, Argon, and Helium-Neon lasers for the detection of various fluorochromes. We can sort into12 by 75 mm culture tubes, 15 ml conical type culture tubes, or a variety of plates such as 96 well culture, or PCR plates.
Becton Dickenson LSR II
This instrument is an analyzer capable of 10 color simultaneous data collection and analysis. The LSR II also has the capacity to do Calcium flux assays which utilize time as a parameter, and is capable of producing a ratiometric parameter of bound, and unbound dye.
BD Biosciences LSR Fortessa
The BD Biosciences LSR Fortessa flow cytometry analyzer has four lasers for fluorochrome excitation, and can detect up to 16 colors simultaneously for multicolor immunofluorescence assays. There is also a HTS available for microtiter well plate sampling.
The current lasers are the Blue (488nm), Violet (405nm), Green (532nm), and Red (633nm).
The sensitivity and resolution of the instrument is equivalent to the LSR II we have now as it uses the same flow cell design, and optical layout
The facility is also equipped with a Miltenyi autoMacs system for the magnetic selection (positive, or negative) of populations of interest in a one step sort or as a presort method of enrichment of a population for subsequent surface staining and selection by FACS of subpopulations with multiple simultaneous surface markers.
Flow Jo iMAC Computer Workstation, plus 2 additional workstations with BD CellQuest & FCAP Array software
An iMAC workstation running FlowJo, CellQuest Pro (BD Trademark), BD FACS DIVA software with use of a MS Windows emulator known as “Parallels”, and FlowLogic analysis software from Inivai, is available for analysis of flow cytometry (FCS) standard format data acquired on any flow cytometer. The workstations also have the standard array of software for producing high quality data graphics output in PDF format suitable for manuscript submissions.
Internal users of the facility are able to move data files collected on the instruments to their own PC’s using a server on the institute’s intranet. Users’ DIVA (bd trademark) experiment templates are also kept and archived indefinitely after periodic compression by facility staff.
Anyone needing a copy of an experiment should contact the data administrator using the following form which can be completed online and emailed.