Assaying Adherent Cells Using Advanced Flow Cytometry
Traditionally, many cell-based flow cytometric assays have been limited to use with suspension cells, without being optimized for use with adherent cells. This is primarily due to the extra step required to lift and dissociate adherent cells to generate a single cell suspension suitable for flow cytometry. Neglecting adherent cells in flow analysis greatly restricts the potential application of many of these assays, since a large number of therapeutic drugs target receptors found on adherent cells particularly in the immuno-oncology area, such as treatments for breast or lung cancers. Therefore in order to achieve the maximum output from flow cytometry, it is essential that during assay development we include the capabilities to interrogate adherent cell types, while avoiding shortfalls of conventional techniques that often:
- Provide bulk measurements (e.g. imaging) without a deeper investigation into effects on cell marker expression or cytokine release
- Require multiple workflows for quantification of different parameters, often necessitating multiple different instruments
- Lack physiological relevance towards in vivo conditions
- Involve lengthy, time-consuming workflows; requiring steps such as protocol optimization, fixation and repetitive washes
Here we present a portfolio of adherent cell assays for the iQue® Advanced Flow Cytometry platform with easy-to-follow protocols for cell lifting and dissociation alongside validated reagent kits and analysis templates. We describe a range of assays across multiple research areas, including analysis of immune cell phenotype and function following killing of adherent tumor cells (in both 2D and 3D models); characterization of drug mechanism of action (MoA) in the context of ADCP and NK ADCC assays and assessment of therapeutic antibody internalization into adherent cell types.
Application
iQue® Adherent Cell Applications
Phenotype & function - Discern immune cell phenotype and function during tumor cell killing in adherent cell models using T Cell Activation, Killing, Exhaustion and Memory kits
MoA characterization - Profile mechanism of cytotoxicity induction with antibodies against solid tumor targets using established workflows and reagent kits
- Antibody-dependent cellular cytotoxicity (ADCC) (NK killing kit)
- Antibody-dependent cellular phagocytosis (ADCP) kit
Antibody interactions - Assess interactions of targeted therapeutic antibodies with live adherent cell types
Advanced cell model analysis - Evaluate immune response to solid tumors using advanced spheroid models
Workflow for lifting adherent cells seeded as 2D monolayers
Figure 1. Easy-to-follow protocol for gentle lifting of adherent cell monolayers for analysis using advanced flow cytometry
Workflow for analysis of immune cells in 3D single spheroid models
Figure 2. Schematic outlining the protocols for analysis of tumor infiltrating lymphocytes (TILs) and immune cell killing (ICK) in 3D advanced solid tumor models using the iQue®
Key Advantages
- Follow simple protocols - Simple cell preparation protocols are combined with mix and read reagent kits to create smooth, easy-to-follow workflows
- Perform high-throughput analysis - The iQue® can acquire a full 96-well plate in only 15 minutes - making adherent cell screening a reality
- Maximize sample usage - Reduce cell loss with concise protocols utilizing gentle dissociation to single cell suspensions while minimizing cell death and maintaining surface marker integrity
- Streamline data acquisition and analysis - Collect and analyze complex data quickly to rapidly drive decisions
