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Peer-reviewed

Multi-color Flow Cytometry Protocol to Characterize Myeloid Cells in Mouse Retina Research

小鼠视网膜研究中骨髓细胞特征的多色流式细胞术方案

WX Wei Xiao *
RS Rami A. Shahror *
CM Carol A. Morris
RC Ruth B. Caldwell
AF Abdelrahman Y. Fouda

 (*contributed equally to this work) 发布: 2023年08月20日第13卷第16期 DOI: 10.21769/BioProtoc.4745 浏览次数: 1763

评审: Vivien J. Coulson-ThomasMunenori IshibashiAnonymous reviewer(s)

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Abstract

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry–based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers.


Key features

• This protocol describes a flow cytometry–based method to analyze the myeloid cell response in retinopathy mouse models.

• The protocol can distinguish between microglia- and monocyte-derived macrophages.

• It can be modified to incorporate markers of interest.

We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.

Keywords: Retinopathy (视网膜病变) search Mouse retina (小鼠视网膜) search Flow cytometry (流式细胞术) search Myeloid cells (髓细胞) search Microglia (小胶质细胞) search Macrophages (巨噬细胞) search

Background

Retinal injury is associated with activation of microglia and infiltration of bone marrow–derived macrophages and other leukocytes. This immune cell accumulation/activation is known to play a vital role in retinal injury progression and repair. While fluorescent immunolabeling coupled with confocal microscopy imaging of retina flatmounts or sections can be used to assess the retinal immune response, it is often limited by the number of antibodies that can be multiplexed, and quantification tends to be tedious and time-consuming due to the need for staining multiple sections. Furthermore, most myeloid cell markers used in imaging studies such as Iba1 and CD68 are expressed by both microglia and macrophages; also, the markers that are thought to be microglia-specific, such as P2RY12 and TMEM119, can be downregulated in activated microglia, thus complicating interpretation of the results (van Wageningen et al., 2019; Honarpisheh et al., 2020). Alternatively, flow cytometry offers a robust quantitative method to analyze immune cell response in retinal injury models. Using flow cytometry, microglia can be distinguished from myeloid leukocytes based on the relative expression of the CD45 marker coupled with the marker CD11b, where microglia are CD11b+/CD45low while myeloid leukocytes are CD11b+/CD45hi. Furthermore, flow cytometers allow for multicolor staining and hence identification of various markers of interest using the same sample. However, conducting flow cytometry on retina tissue is challenging due to the small tissue size and low cell yield. In this methods paper, we describe a step-by-step protocol to obtain high yield of viable cells and analyze different immune cell populations after retina injury. A panel of antibodies is included to identify the immune cell subsets and myeloid cell inflammatory phenotype using proinflammatory (M1-like) marker, CD11c, and the anti-inflammatory (M2-like) marker, CD206. We present representative flow cytometry results from three different retina injury models—retinal ischemia-reperfusion (IR) injury, endotoxin-induced uveitis (EIU), and oxygen-induced retinopathy (OIR)—that model ischemic retinopathy, uveitis, and retinopathy of prematurity, respectively. The IR and EIU are induced in adult mice and represent two different injury modalities, with the latter leading to a stronger immune response. The OIR model assesses the murine pup’s retina response to hypoxia, which involves vascular regression followed by neovascularization.

Materials and reagents

  1. Ketamine 100 mg/mL (Hikma, NDC 0143-9509-01); note that ketamine is a controlled substance and needs a special Drug Enforcement Administration license to be obtained

  2. Xylazine 100 mg/mL (Covertus, catalog number: 1XYL006)

  3. Sodium chloride for injection vial USP (0.9% saline, 50 mL) (Hospira, NDC 0409-4888-06)

  4. Sodium chloride for injection USP IV bag (0.9% saline, 250 mL) (Baxter, NDC 0338-0049-02)

  5. Bovine serum albumin (BSA) (GeminiBio, catalog number: 700-100P)

  6. DNase I (Sigma-Aldrich, catalog number: NC1539905)

  7. Ethylenediaminetetraacetic acid (EDTA), 0.5 M (Thermo Fisher, catalog number: AM9260G)

  8. Dulbecco’s modified Eagle medium (DMEM), high glucose, GlutaMAXTM supplement, pyruvate (Thermo Fisher, catalog number: 10569010)

  9. Fetal bovine serum (FBS) (Thermo Fisher, catalog number: 10438026)

  10. Phosphate buffered saline (PBS, 10×) (Thermo Fisher, catalog number: 70-011-044)

  11. Hank’s balanced salt solution (HBSS) with calcium and magnesium (Thermo Fisher, catalog number: 14025076)

  12. HEPES buffer solution (Millipore Sigma, catalog number 83264)

  13. LiberaseTM (Sigma, catalog number: NC1179175)

  14. Zombie VioletTM Fixable Viability kit (BioLegend, catalog number: 423113)

  15. Jackson Immuno Research Labs normal rat serum (Fisher, catalog number: NC9834724)

  16. RBC lysis buffer for mouse (Thermo Fisher, catalog number: J62150.AK)

  17. FisherbrandTM round-bottom polystyrene test tubes without cap, FACS tubes (Thermo Fisher, catalog number: FB149563A)

  18. Fluorescent conjugated primary antibodies and unconjugated blocking antibodies (CD16/32) (Table 1)


    Table 1. Antibody panel used in this protocol

    AntibodyConc.FluorophoreCloneHostCompanyCatalog #Dilution
    CD11b0.2 mg/mLPerCPM1/70RatBioLegend1012301:100
    CD11c0.2 mg/mLBrilliant Violet 605TM N418Armenian HamsterBioLegend1173341:100
    CD2060.5 mg/mLFITCC068C2RatBioLegend1417041:100
    CD450.5 mg/mLAlexa Fluor® 70030-F11RatBioLegend1031271:100
    F4/800.5 mg/mLPEC1:A3-1RatCedarlaneCL8940PE1:100

    Gr-1 (Ly-6G/

    Ly-6C)

    		0.2 mg/mLAPCRB6-8C5RatBD Bioscience5610831:100
    CD16/CD320.5 mg/mLUsed as (FC block)2.4G2RatBD Bioscience5531421:100


Solutions

  1. Anesthesia cocktail (see Recipes)

  2. Digestion buffer (see Recipes)

  3. Flow cytometry staining buffer (FACS/EDTA buffer) (see Recipes)


Recipes

  1. Anesthesia cocktail

    Mix 2 mL of ketamine (100 mg/mL), 0.2 mL of xylazine (100 mg/mL), and 1.8 mL of 0.9% saline to achieve a final solution of 50 mg ketamine and 5 mg xylazine per 1 mL.

  2. Digestion buffer

    Supplement HBSS with 5% FBS prepared from frozen stock (triple filtered by the manufacturer through a 0.1 μm filter) and 10 mM HEPES, 0.5 mg/mL of liberase, and 0.1 mg/mL of DNase I. Mix gently for 5 min at 4 °C and keep protected from light until needed. Make sure that the FBS used in the digestion buffer is filtered. If not, filter the entire digestion buffer through a 0.22 μm filter to avoid any cellular aggregation that may occur due to potential debris in the unfiltered FBS.

  3. Flow cytometry staining buffer (FACS/EDTA buffer)

    Prepare by adding 1 mL of 0.5 M EDTA to 50 mL of 5% BSA in 1× PBS.

Equipment

  1. BD LSRFortessa flow cytometer; analyzes up to 16 colors (BD Biosciences)

  2. Digital Peri-StarTM Pro peristaltic perfusion pump (World Precision Instruments)

  3. Eppendorf benchtop centrifuges (for 2, 15, and 50 mL tubes at room temperature and 4 °C)

  4. Lab ArmorTM 37 °C bead bath (Fisher)

  5. Invitrogen Countess III cell counter and counting chamber slides (Thermo Fisher)

  6. Bench-top vortex (Fisher)

  7. Dissection tools (World Precision Instruments, catalog number: MOUSEKIT)

  8. General lab supplies: tubes, pipettes, tips, etc.

  9. Insulin syringes (U-30, short needle, 30 gauge) for ketamine/xylazine administration (MHC medical)

  10. Surgical blades, No. 10 (Medline, catalog number: MDS15010)

  11. Weighing balance (Jscale, catalog number: CJ-4000)

  12. 30 G needle (TURMO, catalog number: NN3025R)

  13. 25 G needle (Air-Titn, catalog number: 830003786)

  14. Hemostat (World Precision Instruments, catalog number: 15920)

  15. Styrofoam or wood surgical station

Software

  1. FlowJo (software package for analyzing flow cytometry data), https://www.flowjo.com/

Procedure

English
中文翻译

文章信息

版权信息

© 2023 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).

如何引用

Xiao, W., Shahror, R. A., Morris, C. A., Caldwell, R. B. and Fouda, A. Y. (2023). Multi-color Flow Cytometry Protocol to Characterize Myeloid Cells in Mouse Retina Research. Bio-protocol 13(16): e4745. DOI: 10.21769/BioProtoc.4745.

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分类

神经科学 > 细胞机理 > 小神经胶质

免疫学 > 免疫细胞染色 > 流式细胞术

细胞生物学 > 基于细胞的分析方法 > 流式细胞术

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