利用流式细胞术对巯基乙醇酸盐诱导的小鼠腹腔巨噬细胞中LPS-TLR4复合物内化的定量分析

Katharina Richard; Stefanie N. Vogel; Darren  J. Perkins

doi:10.21769/BioProtoc.3369

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Peer-reviewed

Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal Mouse Macrophages by Flow Cytometry

利用流式细胞术对巯基乙醇酸盐诱导的小鼠腹腔巨噬细胞中LPS-TLR4复合物内化的定量分析

KR Katharina Richard

SV Stefanie N. Vogel

DP Darren J. Perkins email

发布: 2019年09月20日第9卷第18期 DOI: 10.21769/BioProtoc.3369 浏览次数: 4531

评审: Alka MehraMartin V KolevMarieta Ruseva

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Cover of Nature Immunology, featuring study using the protocol.

Dec 2018

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Abstract

Internalization of cell surface Toll-like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.

Keywords: TLR4 (Toll样受体4)

LPS (LPS)

TRIF (β干扰素TIR结构域衔接蛋白)

CD14 (CD14)

Endocytosis (内吞作用)

Peritoneal macrophages (腹腔巨噬细胞)

Background

Toll-like Receptor 4 (TLR4) is unique among the Toll-like receptors in that it can deliver qualitatively different signals from two distinct cellular locations (reviewed in Takeda and Akira, 2004). Cell surface TLR4/MD2 is initially ligated by extracellular bacterial lipopolysaccharide (LPS) and engages the intracellular adaptor MAL and MyD88. Subsequently a fraction of cell surface LPS bound TLR4/MD2 will undergo CD14 and clathrin dependent internalization and translocation to an early endosomal compartment (Zanoni et al., 2011). In this endosomal compartment TLR4, engages the signaling adaptors TRAM and TRIF. Much of the regulation of TLR4 internalization and transport remains to be discovered. A flow cytometric method for quantifying loss of TLR4 surface expression, as a metric for endosomal translocation was first described in Bone Marrow Derived Macrophages (BMDMs) by Jonathan Kagan and colleagues (Kagan et al., 2008). Here we describe a modified version of this protocol to enable quantitation of TLR4 internalization in thioglycollate-elicited peritoneal macrophages on which we have published (Rajaiah et al., 2015; Perkins et al., 2018).

Materials and Reagents

Consumables
1. Pipette tips
2. Ice buckets
3. Pipettes
4. 10 ml syringes (BD, catalog number: 309604)
5. 18 G x 1 needles (BD, catalog number: 305195)
6. 50 ml conical tubes (Denville, catalog number: C1062-P)
7. FACS tubes (round), sterile, with caps (Corning, Falcon, catalog number: 352054)
8. FACS tubes (round), non-sterile, without caps
9. Cell strainers, 70 μm (Fisherbrand, catalog number: 22363548)
10. 0.22 μm filters for filtering culture media and FACS buffer (Sarstedt, catalog number: 83.1823.001)
Reagents
1. Ultra pure LPS, purified from E. coli K235 by the hot phenol method as previously described (McIntire et al., 1967)
2. Unconjugated rat IgG2a, λ anti-mouse CD16/32 (FC receptor) antibody, clone 93 (Biolegend, catalog number: 101302)
3. PE-conjugated anti-mouse CD284 (TLR4) antibody, clone SA15-21 (Biolegend, catalog number: 145404)
4. PE-conjugated Isotype control antibody, Rat IgG2a, κ, clone RTK2758 (Biolegend, catalog number: 400507)
5. Optional: other antibodies for cellular identification (e.g. Alexafluor-conjugated anti mouse F480; Biolegend, catalog number: 123119)
6. Optional: paraformaldehyde solution (PFA), 4% in PBS (Affymetrix, catalog number: 19943)
7. Sterile Saline (Baxter, catalog number: 0338-0048-03)
8. Optional: ACK lysis buffer (Quality Biological, Inc., catalog number: 118-156-721)
9. Sterile Phosphate Buffered Saline (PBS) (Corning, catalog number: 21-040-CV)
10. Culture Media (see Recipes)
  1. RPMI 1640 (Corning, catalog number: 15-040-CV)
  2. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11550)
  3. Penicillin/Streptomycin (Gibco, catalog number: 15140-122)
  4. L-glutamine (Gibco, catalog number: 25030-081)
11. FACS buffer (see Recipes)
  1. Sterile Phosphate Buffered Saline (PBS) (Corning, catalog number: 21-040-CV)
  2. EDTA, 0.5 M (Quality Biological, Inc., catalog number: 351-027-101)
  3. Fetal Bovine Serum (FBS) (Atlanta Biologicals, catalog number: S11550)

Equipment

Light microscope with hemocytometer slide
37 °C incubator with CO₂ control designated for cell culture work (no bacterial/viral work)
Biosafety Cabinet designated for cell culture work (no bacterial/viral work) Sterile Culture Hood
Vortex Genie
Refrigerated Swing Bucket Centrifuge with inserts for 50 ml conical tubes and flow cytometry tubes
Flow Cytometer (e.g., Becton Dickinson LSR II)
Computer for analysis

Software

FCS Express 6.0 Flow (De Novo Software; Glendale, CA USA)
GraphPad Prism 7 (Graph Pad Software; La Jolla, CA USA)

Procedure

English

中文翻译

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Richard, K., Vogel, S. N. and Perkins, D. (2019). Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal Mouse Macrophages by Flow Cytometry. Bio-protocol 9(18): e3369. DOI: 10.21769/BioProtoc.3369.