Suspension Cell Assays

Staining of human bone marrow cells on the DropArray Microplate

Staining of human bone marrow
cells on the DropArray Microplate.

The process of washing suspension cells — pipetting, centrifuging, and dispensing — can be very disruptive and lead to loss of reagents and cells and cause unnecessary strain. In addition, the procedure is overly time consuming and is subject to many error-prone steps. Overall, these limitations make it unfeasible to perform many cellular assays with suspension cells, including high throughput assays.

The DropArray platform lets these cells be retained, allowing entire assays to be performed directly on the proprietary, wall-less microtiter plate — the DropArray Microplate. Its unique design allows researchers to culture as little as 250 cells (384-format) or 750 cells (96-well format) or as many as 10,000 cells (384-well format) or 40,000 cells (96-well format) per assay. In addition, plate washing is performed using gentle, low-velocity liquid exchange that keeps suspension cells within the well. Overall, this enables fully automatable, plate-based assays with suspension cells that were previously impractical.

  • Robust: Up to 90% retention of suspension cells after wash steps
  • Flexible: Expands assay capabilities for suspension cells
  • High-throughput: Allows for large-scale assays to be performed at a fraction of the cost, working volume is 8-20 µl for a 96-well plate and 2-4 µl for a 384 plate
  • Customizable: Can be used with standard pipettes, imagers, and dispensers
  • Reliable: Statistically valid results using fewer scarce or precious cells
Cell Type Application DropArray Platform Advantages
All Suspension Cells Immunofluorescence
FACS
Cell viability assays
High retention of suspension cells after washing is consistent and reproducible
4 - 10 times fewer cells required per data point conserves cells
Compatible with standard liquid dispensers, imagers and readers for microplates
B and T Cells Cytotoxic T-cell killing High retention of B-Cells and T-Cells after washing
Live, real-time monitoring of killing events by individual T-Cells
Conservation of cells by 4-fold per well (data point)
PBMCs Multi-step, wash-based assays Highly efficient (70-95%) retention of PBMCs after washing
Conservation of cells by 4-fold per well (data point)
Blood, Bone Marrow Cells Long-term staining/culture assays Maintain cells in culture for long incubation periods without evaporation
Retain cells upon washing

 

Suspension Cell Washing Assays: PBMCs are Retained After Multiple Steps

Washing suspension cells has always posed a significant challenge for researchers. For cells like PBMCs, the process is tedious and extremely inefficient. Specifically, centrifugation of cells required for washing is time consuming and results in loss of cells, making the process unreliable and inconsistent. Here we demonstrate a series of washing steps performed on PBMCs using the DropArray platform. Wash after wash, the PBMCs are effectively retained on the microplate. In addition, you can see that the PBMCs continue to move around the wall-less well and do not become attached to the surface of the plate.

 

Pre-wash
Read 1: Pre-wash

3X wash
Read 2: 3X wash

6X wash
Read 3: 6X wash

 

 

DAPI superposition of all reads

DAPI superposition of all reads.

Number of cells per field of view after each read with different seeding densities.

Number of cells per field of view after each read with different seeding densities.

 

Performance Review

Previous challenges:
Cellular assays using PBMCs that involve washing steps result in substantial cell loss, leading to potentially unreliable and inconsistent data.

DropArray platform advantages:
Effective retention of PBMCs after washing allows for accurate and consistent data.

 

Immunofluorescence Assays: Novel Assays for Monitoring Cytotoxic T Cell Killing

Cytotoxic T-cell killing is an important assay in immunotherapy development. Bispecific antibodies can be used to stimulate T-cells and trigger tumor cell killing. Previously, FACS was used to monitor these assays on a global scale, although individual visualization of the events remained impractical and time-lapse experiments were non-existent. In addition, B cells and antibodies were used in excess to overcome the loss of materials experienced during washing steps. Here, the DropArray system was used to overcome many of these hurdles to allow for real-time visualization of single cells and quantification of B-cell death from T-cell killing.

CMFDA-labeled B lymphoma, BJAB cells (green) were incubated with the bispecific antibody and anti-CD8–APC-labeled T cells (blue) on the DropArray Microplate. T cell–mediated killing of the BJAB cells is seen as positive PI staining (red) in a green cell adjacent to a blue cell. The various panels (a, b, c) show different types of T-cell killing activities.

 

Performance Review

Previous challenges:
The readout for cytotoxic T-cell killing has been FACS, which provides info on a population of cells, rather than individual cellular events.

DropArray platform advantages:
The DropArray platform allows the multiplex labeling of B- and T-cells, which permits imaging of individual cytotoxic events at the single-cell level. Overall, this labeling and imaging allows for accurate information on the nature of the cytotoxic T-cell killing that was previously impractical.

 

Long-term Incubation: Expanding Assay Timelines and Flexibility

Human bone marrow cells can be hard to obtain in large quantities. In addition, culturing small numbers of cells over long periods can be difficult and lead to cell loss due to evaporation or other inconsistent culture conditions. The DropArray platform allows small numbers of cells to be easily maintained for various cell assays, including those involving bone marrow cells.

Staining of human bone marrow cells on the DropArray Microplate

Post 6-day primary human bone marrow cells were stained for DNA (Hoechst) and CD11B (suspension erythrocyte-like cells).

Performance Review

Previous challenges:
Human bone marrow cells can be limited and are expensive to obtain.

DropArray platform advantages:
Bone marrow cells are highly conserved so minimal amounts of cells per assay point are used, allowing for long term culturing in a cost-effective manner.

 

See how the DropArray platform performs with suspension cultures in our video library.

Review our collaborators publications using the DropArray platform.

Learn more about the benefits of the DropArray system.