Cell isolation procedures

From LipidomicsWiki

In order to correlate cellular function with lipid blood levels blood cells need to be analyzed in parallel. To realize this Cellomics approach standardized cell isolation procedures will be developed in collaboration with Miltenyi Biotec GmbH. Since the currently available reagents for cell isolation either lead to cell activation or loose certain subsets of cells development of novel cell isolation kits will be necessary. In collaboration with Miltenyi Biotec GmbH, the Regensburg group is improving the magnetic bead methods to allow 1. a non-activating negative selection of monocytes or alternatively 2. a positive selection of monocytes with novel non-activating antibodies.

[Figure 96; table 19]

The Miltenyi Biotec Separation System has the components autoMACS Pro Separator, MACS MiniSampler and MACS MicroBeads. The autoMACS Pro Separator is an automated immunomagnetic cell separation system, enables sequential processing of up to six samples with sample cooling option.

[Figure 97]

The system is characterized by an optimized sensor-controlled process, highly flexible cell separation, based on renowned MACS technology, compatible with MACS cell separation
reagents, and guarantees standardized procedure for reproducible results. The autoMACS Pro Separator allows handling of multiple samples with the help of an automated arm and the
new MACS MiniSampler. The MiniSampler can hold one of three different specially designed tube racks for automated loading of sample volumes from 0.2 to 50 mL. The autoMACS Pro Separation technology uses a column placed in the magnetic field of a MACS Separator that creates a high gradient magnetic field strong enough to retain cells labelled with minimal amounts of MACS Microbeasds. The MACS technology allows gentle isolation of viable, functionally active cells, excellent purity and recoveries, thorough rinsing procedure, flexible cell sorting strategies, positive selection, depletion, or subset sorting, manual or automated separation. The MACS MicroBeads are small (50 nm) superparamagnetic particles, coupled to highly specific antibodies, allow short incubation times, are non-toxic and biodegradable. No bead detachment is required: Labeled cells can go straight to flow cytometric analysis, cell culture, or other downstream applications.
RoboSep® (stemcell technologies) may be an alternative to the Miltenyi Biotec Separation system. It combines the versatility of the popular EasySep® technology with the convenience of walk-away automation. Customizable programs allow positive or negative selection of virtually any cell type from any species. Up to four samples can be processed
simultaneously. A major advantage is the fully automated system. Samples & reagents are simply loaded enabling walk away of employees. No pre-labeling is necessary. Processes up to 4 samples simultaneously are possible. It is extremely versatile, uses EasySep® immunomagnetic cell separation technology, no columns are necessary, positive or negative selection can be chosen, it isolates virtually any cell type from any species and sample type - now including whole blood. Tiny magnetic nanoparticles are compatible with flow cytometry analysis. The capacity is 6 x 10⁵ to 8 x 10⁹ cells. Positive selection consists of up to 4 different simultaneous separations; up to 8 x 109 total cells (4 samples of up to 2 x 109 cells each). Negative selection is for up to 2 different simultaneous separations; up to 2 x 10⁹ total cells (2 samples of up to 10⁹ cells each).
Negative and positive selections can be executed simultaneously. Sample volume may be from 0.25 mL to 8.5 mL per sample, or up to 4.5 mL whole blood. Safe sample handling is
safe, compatible with most biocontainment hoods, disposable tips to eliminate crosscontamination are used. The Robo Sep is easy to use as an existing program can be chosen
or an own protocol is customized. Touch-screen operation is possible.

[Figure 98]