A sample preparation for multi-color flow cytometry commonly involves the use of a U-bottom 96 well microtiter plate(MTP) or a flow cytometry tube. An incubation of millions of cells in suspension is performed in the vessel of choice with a cocktail of fluorescently labelled antibodies. The traditional method of removing unbound antibody fraction involves centrifugation. As seen in figure 1A, centrifugation involves 2-4 wash intervals involving multiple steps with significant hands on time. There are inherent issues with centrifugation of suspensions cells:
- Packing/pelleting of cells produces significant stress that can alter cellular biomarker pathways
- Significant (>50%) cell loss can occur
- User-dependent variability and inconsistency from lab to lab are common
DA-Cell solution enables sample preparation for multi- color flow cytometry and bypasses the need of centrifugation for washing suspension cells. The DA-Cell technology is designed to maintain samples in a 96 droplet based format instead of using a conventional microwell with walls (Figure 1B). DA-Cell is a centrifuge-less processing method using the unique wall-less features of the DropArray technology combined with a fully automated washer. The DA-Cell washer generates a laminar flow for each drop on the DA-Cell plate via dual-nozzle action, with one nozzle dispensing liquid into the drop and the other nozzle aspirating liquid. The DA-Cell workflow is superior over the traditional centrifugation method:
- Cell retention >95%
- Clear and easy gating and segregation of cell populations as a result of better washing and cell preservation
- Reduced hands on time with complete washing in 2-4 minutes
- Consistent and reproducible data with elimination of user variability
Figure 1: Comparison of a washing workflow for the preparation of cells suspension for analysis by flow cytometry. Laminar flow washing with DA-Cell is as effective as washing via centrifuge.
We have shown that data generated from DA-Cell centrifuge-less processing and washing results in superior data generated from either microtiter plate or FACS tube using centrifugation. As shown if Figure 2, DA-Cell results in superior data over microtiter plate with improved resolution and higher stain index as well as unbiased clustering and automated gating of cell populations.
Figure 2: Comparison of a DA-Cell versus traditional 96-well microtiter (MTP) plate using centrifugation. PBMCs were aliquoted by 1 million per well for both DA-Cell and MTP. A 21-color mastermix of antibodies was added, mixed, and cells washed by their respective method as shown in Figure 1. Cells were collected from the plate and transferred to a tube for data acquisition by flow cytometry. DA-Cell distinguished cell populations more effectively, resulted in higher stain index, and produced a more accurate heat map for the 21-color assay.
Improved enumeration of γδ T Cells with DA-Cell in whole blood assay
- Better cell retention of live cells
- Higher stain index of VD2 marker (also CD4 and other markers)
- Higher enumeration of rare GDT cells