采用磁珠从树突细胞中分离吞噬体

Bénédicte Manoury

doi:10.21769/BioProtoc.820

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Peer-reviewed

Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads

采用磁珠从树突细胞中分离吞噬体

Bénédicte Manoury email

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.820 浏览次数: 10452

评审: Fanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS Pathogens, featuring study using the protocol.

Aug 2012

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

Abstract

Phagosomes are intracellular organelles in dendritic cells in which pathogens such as viruses, bacteria and parasites are internalised to be proteolysed and killed. Phagosomes are formed by fusion with the plasma membrane, some area of the endoplasmic reticulum as well as the lysosome. This protocol described the purification of phagosomal compartments at different stage of their maturation using magnetic beads.

Materials and Reagents

CO₂ independent medium (Life Technologies, Invitrogen^TM, catalog number: 18045054 )
Iscove’s Modified Dulbecco’s Medium (IMDM) (Sigma-Aldrich, catalog number: I3390 )
Fetal calf serum (Hyclone/PAA, catalog number: sv-143-03 )
Penicillin streptomycin (Sigma-Aldrich, catalog number: P11-010 )
Glutamine (Sigma-Aldrich, catalog number: G7513 )
GM-CSF (10 ng/ml) (Peprotech, catalog number: 315-03 )
EDTA (Life Technologies, Invitrogen^TM, catalog number: 15575-038 )
BSA (Sigma-Aldrich, catalog number: A2153 )
DTT (Sigma-Aldrich, catalog number: 43813 )
ULTRA tablets protease inhibitor cocktail solution (Roche, catalog number: 0 5892791001 )
NaCl (Sigma-Aldrich, catalog number: S-7653 )
Tris pH 7 (Sigma-Aldrich, catalog number: T-1503 )
NP40 (Sigma-Aldrich, catalog number: I8896 )
MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
EGTA (Sigma-Aldrich, catalog number: E3889 )
Imidazole (Sigma-Aldrich, catalog number: I-5513 )
Sucrose (Sigma-Aldrich, catalog number: S79-03 )
RIPA (Pierce Antibodies, catalog number: 89900 )
Antibodies:
EEA1 (Abcam, catalog number: ab2900 )
TfR rabbit serum (Home made)
LAMP1 (Abcam, catalog number: ab62562 )
Rab7 (Abcam, catalog number: ab126712 )
1x PBS (see Recipes)
RIPA buffer (see Recipes) supplemented with 2 mM DTT and 1x complete ULTRA Protease inhibitor cocktail tablets
Bone marrow derived dendritic cells medium (BMDCs) (see Recipes)
Homogenisation buffer (HB) (see Recipes)
Lysis buffer (see Recipes)

Equipment

Water bath
Magnetic beads (Dynal, Dynabeads M-280 streptavidine)
Magnetic stand for eppendorf (Dynal, catalog number: R670001 )
22 g Needle (Terumo, catalog number: NN-2238R )
1 ml Syringe (Terumo, catalog number: S*025E1 )
Centrifuge

Procedure

English

中文翻译

文章信息

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如何引用

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

Manoury, B. (2013). Isolation of Phagosomes from Dendritic Cells by Using Magnetic Beads. Bio-protocol 3(14): e820. DOI: 10.21769/BioProtoc.820.
Maschalidi, S., Hassler, S., Blanc, F., Sepulveda, F. E., Tohme, M., Chignard, M., van Endert, P., Si-Tahar, M., Descamps, D. and Manoury, B. (2012). Asparagine endopeptidase controls anti-influenza virus immune responses through TLR7 activation. PLoS Pathog 8(8): e1002841.