LEAP in situ cell purification offers these benefits:

• Clone or enrich cell populations based on cell phenotype
• Rapid whole-well imaging – brightfield and/or fluorescent analysis
• Image-based validation of purification
• Purify adherent or non-adherent cells
• Purify cells, right where they grow, without creating single cell suspensions
• Purify intact cell colonies without dissociating (e.g., ESCs, iPSCs)
• Consistent high yield and high purity
• Purify small samples (10's - 10⁶)
• Laser processing is done with the microplate lid in place:
• reducing risk of contamination
• suitable for biohazardous samples

Efficient laser processing on LEAP requires C-lect plates which have:

• high optical quality for even illumination, imaging, and segmentation
• consistent thickness, transmission, and refractive index for optimal laser performance
• SBS-standard foot print with proper well definition to ensure full well processing

We offer the following C-lect plates for LEAP processing:

Visit C-Lect Consumables

We continue to expand our plate offerings. New plate types available in June 2011:

• 1-well Stem Cell Plate
• 24-well Stem Cell Plate
• 48-well Stem Cell Plate

Efficient laser processing on LEAP requires C-lect plates which have:

• high optical quality for even illumination, imaging, and segmentation
• consistent thickness, transmission, and refractive index for optimal laser performance
• SBS-standard foot print with proper well definition to ensure full well processing

Only C-lect plates can be used for laser processing.

The use of non C-lect plates for cell analysis only is currently in development.

Plate processing times vary due to: 1) application 2) cell number 3) plate type

Range of processing times per plate:

Improvements to decrease plate processing times are currently in development.

Cells are eliminated on LEAP by one of three methods:

• Photothermal: Rapid cell necrosis using the green laser & PhotoTherm reagent
• Photomechanical: Cell lysis using the green laser
• Photochemical: Induction of apoptosis using the UV laser

LEAP has 2 lasers. Choice of the laser depends on the particular application and experiment you are performing.

Typically:

• Green laser (532 nm): Cell Purification, Stem Cell Passage, EB Generation, & Stem Cell Colony Purification
• UV laser (355 nm): Cell Purification, Cell Secretion

PhotoTherm is a non-toxic FDA approved dye that is added to the cell culture medium to absorb the energy of the laser, causing specific photothermal necrosis of cells.

PhotoTherm Reagent has no effect on growth or viability of primary cells or cell lines.

PhotoTherm Reagent has no effect on growth, morphology, or pluripotency of ESCs or iPSCs.

Laser processing only affects the cells that are shot by the laser, causing them to be eliminated.

Cells that are not selected for purification/processing will not be touched by the laser and thus will not be affected.

Importantly, the desired cells are subjected to far less stress than conventional purification techniques which require significant manipulation of all cells (e.g., trypsinization, dissociation, filtration, sorting or bead attachment, etc.)

We and our customers have purified a wide variety of cell types with high purity and high yield including many different cell lines, primary cells, stem cells, and stem cell-derived cell types.

Different lines of this specific cell type have been purified/processed on LEAP

Laser processing is extremely focused and thus does not produce detectable temperature changes in the well or medium.

The LEAP lasers produce extremely short (<1 ns) pulses to purify/process cells, which does not cause temperature changes in the well or medium

Char is a small black mark on the plate bottom that is caused by laser shooting when the laser focus is very close to the plate bottom.

Char can be minimized by:

• Increasing the laser spot size (beam expander)
• Decreasing the shooting stage Z offset

The optimal dye for live cell staining will depend on cell type.

All cell dyes should be tested to: 1) identify the lowest dye concentration with good signal-to-noise 2) determine the cell viability after staining

Recommended dyes & concentration range:

The optimal plating density will vary due to: 1) application 2) cell type 3) plate type

Recommended plating densities:

1 brightfield channel

3 fluorescent channels

• Blue: 385 nm LED (377/50 excitation, 447/60 emission filters)
• Green: 464 nm LED (483/32 excitation, 536/40 emission filters)
• Red: 528 nm LED (531/40 excitation, 629/56 emission filters)

Fluorescent channels can be imaged individually or in combination

Fluorescent channels can be combined with brightfield

Numerical data files are formatted as comma separated value (csv) files

Images may be generated in bmp, jpeg and tiff formats

Data files may be exported in FCS or Multiple FCS for use in flow cytometry software such as FlowJo and FCS Express

1 channel datasets are approximately 1.5GB per plate. Accordingly, a 2 channel experiment would require ~3GB. This is generally independent of plate type as one 96 well contains 16 fields/well for a total of 1,536 images and one 384 well contains 4 fields/well for the same total of 1,536 images.

Numerical data is exported in a comma separated value (csv) format for analysis in programs like Excel, GraphPad Prizm and OpenOffice.org Calc.

Image files may be analyzed with any software that accepts bmp, jpeg or tiff files (MetaMorph, Image-Pro Plus, CellProfiler).

Data files may be exported in FCS or Multiple FCS for use in flow cytometry software such as FlowJo and FCS Express.

Cyntellect offers a Celigo Satellite Workstation that can be used for offline processing of Celigo data.

No, the Celigo does not have network database functionality at this time.

Manual registration- This method of registering the focus allows the user to quickly define the best focus for a given scan manually. The registration is not saved and therefore cannot be loaded for subsequent scans.

Auto registration – The software automatically determines the focus registration using an image-based algorithm (which may be adjusted manually using an offset). Using this method the focus registration is stored and may be used for batch analysis.

Cyntellect offers an automation application programming interface (API) for the Celigo that enables automated plate loading, scanning and processing using a robotic arm such as the Plate-Crane or Hamilton Star.

Accurate segmentation is dependent on exposure and focus settings that give the best contrast.

In general, cells with larger amounts of cytoplasm, such as HeLa’s segment better using a "bright focus" and those that are flatter with less cytoplasm (epithelial cells) may require a "dark focus". Please see the Celigo Users Guide for details on each focus type.

Independent: Each channel is segmented and processed separately. Subpopulations of cells may be present in one channel and absent in another, therefore, data is only acquired for cells in channels where they emit a positive signal. Negative cells are not included in the analysis.

Mask: Data is acquired in all channels for cells detected and segmented in the “Mask” channel only. If the entire population possesses the “Mask” marker, all cells will be included in the analysis.

Merge: Each channel is segmented separately and processed separately. The objects overlays are then merged to create a single mask. Cells segmented in different channels are processed with this merged single mask to include all cells in the analysis even if they have signal in only one channel.

At 3.5X magnification with 1um/pixel resolution, some organelles cannot be resolved on the Celigo. However, nuclei and cytoplasm of most cell types can be segmented and analyzed allowing expression comparisons between these sub-cellular compartments.

There is no current method of counting colonies on the Celigo.

The large imaging area of Celigo is ideal for imaging small organisms; however, the analytical software is not currently available.