吞噬实验检测骨髓来源树突状细胞对坏死肿瘤细胞的摄取

Tania Løve  Aaes; Dmitri V. Krysko; Peter  Vandenabeele

doi:10.21769/BioProtoc.1997

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Peer-reviewed

Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs

吞噬实验检测骨髓来源树突状细胞对坏死肿瘤细胞的摄取

Tania Løve Aaes

DK Dmitri V. Krysko

PV Peter Vandenabeele email

发布: 2016年11月05日第6卷第21期 DOI: 10.21769/BioProtoc.1997 浏览次数: 12622

评审: Xi FengJalaj GuptaRuth A. Franklin

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Cover of Cell Reports, featuring study using the protocol.

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Abstract

This protocol is a flow cytometry-based method to measure the phagocytosis efficiency of necroptotic target cells by bone marrow-derived dendritic cells (BMDCs) in vitro (Aaes et al., 2016). The method is a slightly modified and updated version of the protocols used in previously published papers (Krysko et al., 2006; Brouckaert et al., 2004). In brief, the target cells are labeled with a CellTracker^TM dye before they are induced to undergo cell death. After a co-culture period of 2 h with BMDCs, the cells are immunostained with a dendritic cell marker and dead cell marker, and the phagocytic efficiency is quantified using a flow cytometer. This protocol can readily be used for target cells undergoing cell death modalities other than necroptosis.

Background

Studying the phagocytic uptake of necroptotic cells by BMDCs in vitro, is a preliminary step in the examination of immunogenic cell death models (Obeid et al., 2007). Efficient uptake will allow the phagocyte to cross-present antigens to leukocytes and thereby create an immune reaction towards the dead target cells. In this protocol we make use of a CellTracker^TM dye. This type of dye may be toxic in certain concentrations, which may vary depending on which cell type is used. Thus, we recommend users to first find the optimal concentration of the dye for the target cell in use. Optimally, the CellTracker^TM dye itself should not induce any cell death, but should label the target cells so that they become easily separable from the CD11c-positive BMDCs.

Materials and Reagents

Delta treated 10 cm Petri dishes (Thermo Fisher Scientific, Nunc^TM, catalog number: 153066 )
15 ml polystyrene centrifuge tubes (Corning, Falcon^®, catalog number: 352095 )
6-well suspension plates (SARSTEDT, catalog number: 83.3920.500 )
96 V well, 2.0 ml polypropylene plate (Greiner Bio One, MASTERBLOCK^®, catalog number: 780285 )
5 ml round-bottom polystyrene tubes (Corning, Falcon^®, catalog number: 352054 )
Bone marrow-derived dendritic cells (BMDCs) isolated from BALB/c WT mice (see Procedure)
CT26 Necroptosis-inducible cells (DD_RIPK3) (Aaes et al., 2016)
Dulbecco’s modified Eagle medium (DMEM), high glucose (Thermo Fisher Scientific, Gibco^TM, catalog number: 41965062 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10270106 )
Sodium pyruvate (Sigma-Aldrich, catalog number: S8636 )
L-glutamine (Lonza, catalog number: BE17-605F )
Roswell park memorial institute (RPMI) 1640 medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 52400025 )
2-mercaptoethanol (50 mM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 31350010 )
Murine GM-CSF (VIB Protein Service Facility, UGent-VIB Inflammation Research Center)
ACK lysing buffer (Lonza, catalog number: 10-548E )
Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14190094 )
CellTracker^TM green CMFDA dye (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C7025 )
Doxycycline (Sigma-Aldrich, catalog number: D9891 )
B/B homodimerizer (Takara Bio, catalog number: 635059 )
Purified rat anti-mouse CD16/CD32 (Mouse BD Fc Block^TM) (BD, Pharmingen^TM, catalog number: 553142 )
APC hamster anti-mouse CD11c (BD, Pharmingen^TM, catalog number: 550261 )
SYTOX^® blue nucleic acid stain (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: S11348 )
BD FACSuite CS&T Research Beads Kit (BD, FACSuite^TM, catalog number: 650621 )
DMEM CT26 culture medium (see Recipes)
RPMI BMDC/Co-culture medium (see Recipes)
FACS buffer (see Recipes)
Staining solution (see Recipes)

Equipment

Table top centrifuge
BD FACSVerse^TM flow cytometer (BD, model: BD FACSVERSE )

Software

BD FACSuite^TM software (BD)
FlowJo software version 10.0.8 or newer (FlowJo)

Procedure

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如何引用

Aaes, T. L., Krysko, D. V. and Vandenabeele, P. (2016). Phagocytosis Assay to Measure Uptake of Necroptotic Cancer Cells by BMDCs. Bio-protocol 6(21): e1997. DOI: 10.21769/BioProtoc.1997.