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Intravital Imaging of Intestinal Intraepithelial Lymphocytes

肠上皮内淋巴细胞的活体成像

SM Sara McArdle
GS Goo-Young Seo
MK Mitchell Kronenberg
ZM Zbigniew Mikulski

发布: 2023年07月20日第13卷第14期 DOI: 10.21769/BioProtoc.4720 浏览次数: 3061

评审: Alessandro DidonnaBrahma MuluguMartin V Kolev

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Jul 2022

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Abstract

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

Keywords: Intravital microscopy (活体显微镜) search Intraepithelial lymphocyte (上皮内淋巴细胞) search Tracking (追踪) search Cell labeling (细胞标记) search Suction ring (吸引环) search Intestine (肠) search Confocal reflection (共聚焦反射) search

Background

Intestinal intraepithelial lymphocytes (IEL), sometimes also known as intraepithelial T cells, are one of the largest populations of T lymphocytes in the body. They are located within the epithelium and interact extensively with intestinal epithelial cells by actively patrolling the basement membrane and migrating into the lateral intercellular space (Edelblum et al., 2012; Wang et al., 2014; Hoytema van Konijnenburg et al., 2017; Sumida et al., 2017). IEL are important for the maintenance of integrity of the intestinal barrier, repair of wounds, and protection from pathogenic invasion (Cheroutre et al., 2011). They include innate lymphoid cells but are mostly T lymphocytes.

In previous work, we focused on the role of herpes virus entry mediator (HVEM) as a regulator of CD8α+ intraepithelial T cells in the small intestinal (SI) epithelium. We used intravital imaging to track lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice (Edelblum et al., 2012; Wang et al., 2014). In the previous work, by using mice with deficiency for HVEM expression exclusively in the intestine epithelium we showed that in the SI, epithelial HVEM expression is required for the normal patrolling movement of CD8α+ IEL. This correlated with a decreased response to bacterial infection (Seo et al., 2018). Here, we describe a protocol to analyze CD8α+ IEL in the SI epithelium using intravital imaging.

Materials and reagents

  1. #1.5 12 mm round coverslips (Electron Microscopy Sciences, catalog number: 72230-01)

  2. 31G 0.5 mL insulin syringe for retroorbital (RO) and anesthesia injection (BD, catalog number: 328447)

  3. Vacuum grease, MOLYKOTE® High Vacuum Grease (McMaster-Carr, catalog number: 2966K52)

  4. 10 mL syringe (BD, catalog number: 303134)

  5. Lab tape (Fisher Scientific, catalog number: 15-950)

  6. Antibodies: anti-CD8α-AF488, clone 53-6.7 (eBioscience, catalog number: 53-0081-82); anti-CD8α-AF647, clone 53-6.7 (BD Biosciences, catalog number: 557682); anti-EpCAM-AF647, clone G8.8 (BioLegend, catalog number: 118211)

  7. Sterile phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 10010023)

  8. Isoflurane (Covetrus, National Drug Code: 11695-6777-2, 250 mL, catalog number: 029405)

  9. Oxygen (100% compressed oxygen gas)

  10. Ketamine, Ketaset, 100 mg/mL (Zoetis, National Drug Code: 54771-2013-1)

  11. Xylazine, XylaMed, 100 mg/mL (VetOne, National Drug Code: 13985-612-50)

  12. Vaseline (Fisher Scientific, catalog number: 17-986-496)

  13. 70% isopropanol (Covetrus, National Drug Code: 11695-2178-6, reorder number 002498)

  14. Mice on a C57BL/6 background, available from Jackson Laboratories, kept in specific pathogen-free conditions. We used Hvemflox-neo/flox-neo and Hvemfl/fl mice (Seo et al., 2018)

Equipment

  1. Animal heating controller and 15 cm × 10 cm heating plate (World Precision Instruments, catalog number: ATC2000 and 61830)

  2. Rodent rectal temperature probe (World Precision Instruments, catalog number: RET-3)

  3. Isoflurane vaporizer and mouse isoflurane mask

    Link7 (Patterson Scientific, catalog number: 78919043)

    Uniflow Single Manifold (Patterson Scientific, catalog number: 78924212)

  4. Surgical scissors for skin, small surgical scissors, surgical forceps, Dumont #5/45 forceps

  5. Hair clippers (Wahl, catalog number: 88420) or Nair hair remover sensitive formula

  6. Thermal Cauterizer Unit (Geiger Instruments, model: 150-I or similar unit)

  7. Suction ring assembly

    1. CNC-machined suction ring (parts Prototype Master, Angle Arm, Post, and Extension from Zera Development Co., n.d., Santa Clara, CA)

    2. Indicator swivel clamp with T-handle adjustment, 1/2” diameter × 3/8” diameter (McMaster-Carr, catalog number: 5148A28)

    3. Polyethylene 160 tubing fitted with blunted 18G needle (Warner Instruments, catalog number: 64-0755)

    4. Male Luer lock to 1/4” barb connector (Amazon, catalog number: B0BB649WM2)

    5. Thick-walled vacuum tubing, internal diameter 3/8” or 9.7 mm (VWR, catalog number: MFLX06404-36)

    6. Thick-walled vacuum tubing, internal diameter 5/16” or 7.9 mm (VWR, catalog number: MFLX06404-35)

    7. Vacuum waste gate [can be repurposed from 2-way stopcock (Amazon, catalog number: B09P8GNP7S) or aquarium pump control valve (Amazon, catalog number: B08LGHWB77)]

    8. 5/16” T-splitter (usplastic.com, catalog number: 62095)

    9. Small vacuum trap with fittings for 7.9 mm ID tubing (SP Bel Art, catalog number: F19919-0000)

    10. 1/4” NPT female to 3/8” barb connector (Amazon, catalog number: B09Q813WN1)

    11. Coarse vacuum adjustment valve: 1/4” male-male NPT Valve (Amazon, catalog number: B07YFS1WFX)

    12. 1/4” Barstock tee female adapter (Amazon, catalog number: B07MTWH2W9)

    13. 2” male-male extension nipple (Amazon, catalog number: B000BOA2V2)

    14. 1/4” NPT female-female coupler (Amazon, catalog number: B09651GMQ3)

    15. Vacuum dry pressure gauge, lower mount, 1/4” NPT (Carbo Instruments, catalog number: D25-CSL-V00)

    16. 1/4” NPT male to 5/16” barb connector (Amazon, catalog number: B07RKM6VR8)

    17. Teflon tape (Amazon, catalog number: B095YCMHNX)

    18. Hydrophobic filter, Millex-FG, 0.20 μm, hydrophobic PTFE, 50 mm (Millipore, catalog number: SLFG85010)

  8. Upright Leica SP5 or SP8 confocal microscope or equivalent

    1. Resonant scanner

    2. 25× (0.95) water-dipping, coverslip-corrected objective (Leica 506375)

    3. Piezo Z controller (Piezosystem Jena, catalog number: 0-350-01 or similar)

    4. 488 and 638 nm laser lines

    5. Motorized Movable Base Plate or equivalent for support of the animal stage (Scientifica, model: MMBP-7200-00)

Software

  1. Leica SP8 software LAS X

  2. Fiji (Schindelin et al., 2012) with the Bio-formats and MISTICA Image Alignment (Ray et al., 2016)

    A version of Fiji packaged with MISTICA can be found at github.com/saramcardle/IEL. Otherwise, use the latest Fiji version.

  3. Imaris (Bitplane, version 9.4 or higher)

  4. MATLAB (Mathworks, version 2021a) or the compiled SpiderPlot application (github.com/saramcardle/IEL)

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

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. McArdle, S., Seo, G. Y., Kronenberg, M. and Mikulski, Z. (2023). Intravital Imaging of Intestinal Intraepithelial Lymphocytes. Bio-protocol 13(14): e4720. DOI: 10.21769/BioProtoc.4720.
  2. Seo, G. Y., Takahashi, D., Wang, Q., Mikulski, Z., Chen, A., Chou, T. F., Marcovecchio, P., McArdle, S., Sethi, A., Shui, J. W., et al. (2022). Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection. Sci Immunol 7(73): eabm6931.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 粘膜免疫学 > 上皮

细胞生物学 > 细胞成像 > 活细胞成像

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