Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
流式细胞术检测T淋巴细胞中LFA-1的再循环
发布: 2018年12月05日第8卷第23期 DOI: 10.21769/BioProtoc.3104 浏览次数: 5415
评审: Ralph Thomas BoettcherPiyali SahaRAMESH KUDIRA
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参见作者原研究论文
The authors used this protocol in:
Dec 2017
Abstract
To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow cytometry-based assay that allows the quantification of LFA-1 internalization and its re-expression on the cell surface in T lymphocytes. An advantage of using flow cytometry-based assay over biochemical methods is the low number of needed cells. This protocol can be also used to measure recycling of other receptors.
Keywords: Flow cytometry (流式细胞技术)
Receptor (受体)
LFA-1 (LFA-1)
Endocytosis (内吞作用)
Exocytosis (胞吐作用)
Background
Leukocytes need to be quick to extravasate from the vascular in order to defeat invading pathogens. To become effector cells, T lymphocytes need to migrate in the lymph nodes where they can encounter their specific antigen (Ley et al., 2007). LFA-1, which is the major integrin used by T lymphocytes to adhere and migrate, binds to its ligand intercellular adhesion molecule-1 (ICAM-1) on the endothelial or the antigen presenting cell (Evans et al., 2009). The reuse of LFA-1 is a dynamic process as it is internalized and intracellularly transported to be re-exposed to a new sight on the cell membrane for the cell to move forward (Svensson et al., 2012). The lysosomotropic amine Primaquine can be used to block intracellular transport and when used in T cells LFA-1 dependent migration is impaired (Stanley et al., 2012). The exact mechanism how LFA-1 is internalized and recycled isn’t fully understood. One method to investigate the internalization and re-exposure of LFA-1 in low number of cells is to label cells with the non-blocking antibody for LFA-1 (TS-2) and analyze the internalization and re-exposure of LFA-1 at different time points by flow cytometry (Samuelsson et al., 2017).
Materials and Reagents
- Plastic wares
- Pipette tips (2 μl, 20 μl, 200 μl and 1 ml)
- 96-well polystyrene round-bottom microwell plates (Thermo Fisher Scientific, Nunc, catalog number: 12-565-214)
- Microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001)
- 15 ml conical tubes (SARSTEDT, catalog number: 62.554.502)
- Cells (here we use human primary T lymphoblasts purified from whole blood. Other cell types can also be used)
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030)
- Primaquine diphosphate (PQ) (Sigma-Aldrich, catalog number: 160393)
- Paraformaldehyde, 16% w/v aq. soln., methanol free (VWR, catalog number: 43368.9M)
- Buffers
- Phosphate buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 14040133)
- Hanks balanced salt solution with or without Ca2+/Mg2+ (HBSS) (Thermo Fisher Scientific, Gibco, catalog numbers: 14025092 and 14175095)
- HEPES (Thermo Fisher Scientific, catalog number: 15630080)
- Antibodies
- LFA-1 antibody: Antibody purified from hybridoma TS2/4.1.1 (TS2/4.1.1, ATCC, catalog number: HB-244)
- Secondary Alexa Fluor® 647 donkey anti-mouse IgG (H+L) (Thermo Fisher, catalog number: A-31571)
Equipment
- Pipettes (0-2.5 μl, 2-20 μl, 20-200 μl, 100 μl-1 ml)
- Incubator
- Swing-out Centrifuge for microplates
- Flow cytometry (BD Biosciences, model: LSR II)
Software
- Flowjo, LLC (software for cytometry analysis)
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Potrzebowska, K., Lehtonen, J., Samuelsson, M. and Svensson, L. (2018). Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes. Bio-protocol 8(23): e3104. DOI: 10.21769/BioProtoc.3104.
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分类
免疫学 > 免疫细胞染色 > 流式细胞术
细胞生物学 > 细胞染色 > 蛋白质
生物化学 > 蛋白质 > 免疫检测
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