Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence

Author:

Freeburne Emily1,Teague Seth2,Khan Hina1,Li Bolin1,Ding Siyuan3,Chen Bohan3,Helms Adam4,Heemskerk Idse12356

Affiliation:

1. Department of Cell and Developmental Biology University of Michigan Medical School Ann Arbor Michigan

2. Department of Biomedical Engineering University of Michigan Ann Arbor Michigan

3. Department of Computational Medicine and Bioinformatics University of Michigan Medical School Ann Arbor Michigan

4. Division of Cardiovascular Medicine University of Michigan Ann Arbor Michigan

5. Center for Cell Plasticity and Organ Design University of Michigan Medical School Ann Arbor Michigan

6. Department of Physics University of Michigan Ann Arbor Michigan

Abstract

AbstractDuring development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co‐localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA‐seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi‐well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Iterative immunofluorescenceBasic Protocol 2: Computational analysis pipelineSupport Protocol 1: Generating micropatterned multi‐well slidesSupport Protocol 2: Differentiation of 2D gastruloids

Funder

National Institute of General Medical Sciences

Publisher

Wiley

Subject

Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience

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