Making Deep Immunophenotyping Accessible: The successful application of a guided 23-parameter mouse immunophenotyping panel package provided through a shared resource

Madison G Isbell¹Alex Wendling¹Xinyan Pei¹AMIT KUMAR¹Padmanabhan Mannangatti¹Bradley Krisanits¹Stanley Cheatham¹Marie Michenkova¹Kirill Shumilov¹Rachel G Mendoza¹Matthew E Fernandez¹Allyn Bryan²Thuy-An Nguyen¹Lauren May¹Swadesh Das¹Victoria J Findlay¹Hamid I Akbarali¹Maria Garcia-Bonilla¹David D Limbrick¹Douglas H Sweet¹Sandro R.P. da Rocha¹Alexander Neuwelt^1,2Paul B Fisher¹Devanand Sarkar¹Xiang-Yang Wang¹Joseph William Landry¹Rebecca Kelley Martin^1*

• ¹Virginia Commonwealth University, Richmond, United States
• ²Richmond Department of Veterans Affairs, Richmond, United States

This 23-color mouse immunophenotyping panel was designed and developed by the Virginia Commonwealth University's (VCU) Flow Cytometry Shared Resource (FCSR) to easily bring new use to our high parameter spectral cytometers. Our method is broadly applicable to multiple tissue types, is modifiable, and provides a reproducible, cost-effective option for utilizing high-parameter flow cytometry. To facilitate the mouse immunophenotyping panel, researchers can be provided with optimized reagents, a step-by-step staining protocol, instrument training, pre-run single color controls as well as acquisition and analysis templates to streamline the workflow. Data analysis is generally done with a traditional manual gating strategy but t-stochastic neighbor embedding (tSNE) and uniform manifold approximation projection (uMAP) generation can be performed, as desired. In an FCSR, this panel requires only light preparation work for shared resource (SR) staff with maximum benefit for researchers. Overall, this publication describes how SR facilities can provide additional benefits and services to their clientele by reducing costs, increasing reproducibility, and lowering the barriers of entry for researchers into the field of high parameter spectral flow cytometry. The panel described is used as an example of the application of the included methods, as well as a complete resource for other institutions to utilize themselves.