Microglial Activation Antibody Toolkit: High-Content Methods to Study TREM2 Signaling

Selecting antibodies for molecular signaling research in neurodegeneration is a complex and nuanced process, yet it is essential for advancing our understanding of brain function and disease. This blog explores a collaborative project between CST and FUJIFILM Cellular Dynamics, where we used iCell Microglia products alongside a toolkit of key antibodies to characterize signaling events in neurodegenerative diseases. The antibodies we selected provide a valuable, high-content method for verifying the identity and functionality of iPSC-derived microglia (iMGL), particularly in the context of neuroinflammation.

Read on to learn about the project, or use this link to jump to a list of the human-reactive IF-validated antibodies we suggest for studying microglial activation in neurodegenerative diseases. You can also download the research poster below, which my colleagues and I presented at the Society for Neuroscience (SfN) 2024.

Modeling Human Neuroinflammation Using iPSC-Derived Microglia

Microglia, the brain’s resident immune cells, are central to understanding neuroinflammation and neurodegeneration and have increasingly been recognized for their role in Alzheimer’s disease (AD) and aging. To date, mouse models have been the prevailing method for studying these intriguing cells. However, while mouse and human microglia cells have many similarities, key differences in gene expression that arise, especially during aging, call into question the relevance of mouse models for translational research.¹

Blog author Virginia Bain, PhD, presented the results of this at the Society for Neuroscience (SfN) 2024. 

Human-induced pluripotent stem cells (hiPSCs) can help bridge this gap, enabling the in vitro study of cells that function like human microglia. We chose iMGLs, in particular, to study neuroinflammation because these cells emulate human microglial behavior, making them invaluable for characterizing inflammatory responses.² Their compatibility with 96-well plate formats enables high-content analysis of multiple proteins, enabling a more complete understanding of human microglia activation.

High-Content Immunofluorescence for Microglia Characterization

There are many approaches for characterizing disease-associated microglia and their response to stimuli in vitro. Working in a 96- or 384-well plate format lends itself well to assays measuring protein levels, metabolic function, and morphology, while also evaluating the impact of treatments or genetic manipulation. We chose immunofluorescence, and, in particular, high-content analysis (HCA), for this work because of the ability to simultaneously study protein level, localization, and morphological changes induced by treatments. This approach generates a wealth of data that can be visualized in interesting ways to better understand population dynamics.

Inducing Neuroinflammatory Response

To induce inflammation, we took two separate approaches. First, we explored the classical inflammatory response using lipopolysaccharide (LPS) treatment. LPS treatment is a well-established method in the literature for inducing inflammation, and it is effective with many types of immune cells. 

Of course, LPS is not frequently found in the brain, especially in neuroinflammatory diseases such as AD, multiple sclerosis (MS), and Parkinson’s disease (PD). However, pro-inflammatory cytokines are naturally present and can be introduced in an in vitro system. Our second approach for inducing neuroinflammation used the pro-inflammatory cytokines IFNγ and TNFα, a method that is supported in the literature for use in rat microglia.³

We compared the results of these different approaches and found some surprising differences in cellular morphology.

Antibody Toolkit for Studying Neuroinflammatory Response

1. NF-κB p65

To confirm the induction of inflammation, we stained cells with NF-κB p65 (D14E12) XP^® Rabbit mAb #8242. NF-κB p65 is a powerful readout because it allows you to measure cytoplasmic to nuclear translocation in responding cells. This is a classic approach for HCA because it is possible to cleanly define what is nuclear and non-nuclear signal, quantify it, and look at intensity differences in both the cytoplasm and nucleus of each cell.

We generated lots of data using this method, an example of which is shared below. In the figure, the increase in signal in the nuclei (outlined in white) in cells treated with LPS (right) confirms the induction of inflammatory response.

IF analysis of NF-κB p65 (D14E12) XP^® Rabbit mAb #8242 (green) and CD45 (Intracellular Domain) (D9M8I) XP^® Rabbit mAb (Alexa Fluor^® 555 Conjugate) #62267 (red). Nuclei were stained with DAPI #4083 (not shown) and have been outlined in white to show increased signal with LPS treatment (right) compared to untreated iCell microglia (left).

2. Intracellular CD45

A protein tyrosine phosphatase expressed on the surface of immune cells, CD45 is a reliable marker to use with microglia because this protein acts as a negative regulator of microglial activation by dampening pro-inflammatory responses.⁴ With membrane-bound CD45 labeling all microglia, we were able to use Cell Profiler, a free and open source software for image analysis, to define interesting features about the microglia such as cell size and shape. 

We used CD45 (Intracellular Domain) (D9M8I) XP^® Rabbit mAb (Alexa Fluor^® 555 Conjugate) #62267 as a counterstain on every cell. One of the most fascinating—and unexpected—outcomes of this study was the morphological changes we observed. We found that the LPS-treated microglia were slightly smaller and rounder than the healthy cells, and that the microglia treated with pro-inflammatory cytokines were larger and flatter than the healthy cells. These unexpected findings are worth noting for anyone looking to study neuroinflammation using iMGLs, and who are considering different strategies to induce inflammation.

Related Blog: Simplify iPSCs Research: Antibodies to Identify Pluripotency Markers

If you’re wondering how we multiplexed rabbit host primary antibodies with rabbit host conjugates, check out my previous blog post, which offers some IF multiplexing tips.

3. Phospho-Syk (Tyr525/526)

TREM2 is a cell-surface (triggering) receptor expressed on myeloid cells—in particular microglia, osteoclasts, and subsets of macrophages—that helps modulate inflammatory and immune responses.⁵ When activated, TREM2 forms a complex with DAP12, which leads to a cascade of intracellular signaling events.

Once activated, DAP12 recruits protein tyrosine kinase syk. Phosphorylation within the syk activation loop (Tyr 525 and 526) can be used as a reliable readout of TREM2 signaling.⁶

IF analysis of TREM2 (E4F5G) Mouse mAb #29715 (green), DAP12 (E7U7T) Rabbit mAb #97415 (red), and DAPI #4083 (blue) in AHN iCell microglia.

Some researchers find it challenging to use phospho-specific antibodies for immunofluorescence because there can be a limited pool of phosphorylated protein available. However, in the right system, and with the right antibody, high-quality images are attainable. We generated powerful data and observed clear activation with Phospho-Syk (Tyr525/526) (C87C1) Rabbit mAb #2710 when comparing inflammatory microglia to apparently healthy normal (AHN) microglia. 

TREM2シグナル伝達経路

View the full, interactive pathway on the CST website.

Antibody Tools for Microglia Characterization

We continue to develop a comprehensive portfolio of monoclonal antibodies to further characterize disease-associated cellular processes to understand the cellular changes in microglial activation in neurodegenerative diseases such as AD and PD. While there are many ready-to-ship monoclonal antibodies you can use to characterize microglia using IF, the human reactive ones we recommend are listed in the table below.

In addition to curating an antibody toolkit for this study, we were also interested in seeing what happened to the TREM2/DAP12 complex during inflammation. The research we conducted offers insights into how modulating TREM2-dependent microglial activation—either by upregulating or downregulating this pathway—could help elucidate AD pathology. But, you have to check out my poster to learn more!

参考文献：

1. Human and mouse microglia look alike, but age differently | ALZFORUM. (2017, July 18).
2. Abud EM, Ramirez RN, Martinez ES, et al. iPSC-Derived Human Microglia-like Cells to Study Neurological Diseases. Neuron. 2017;94(2):278-293.e9. doi:10.1016/j.neuron.2017.03.042
3. Lively S, Schlichter LC. Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10). Front Cell Neurosci. 2018;12:215. Published 2018 Jul 24. doi:10.3389/fncel.2018.00215
4. Tan J, Town T, Mullan M. CD45 inhibits CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK pathway. J Biol Chem. 2000;275(47):37224-37231. doi:10.1074/jbc.M002006200
5. Colonna M. The biology of TREM receptors. Nat Rev Immunol. 2023;23(9):580-594. doi:10.1038/s41577-023-00837-1
6. Huang Q, Chan KY, Lou S, et al. An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery. Preprint. bioRxiv. 2023;2023.12.20.572615. Published 2023 Dec 22. doi:10.1101/2023.12.20.572615