采用体外分析法检测细胞死亡后的钙网蛋白暴露、ATP和HMGB1释放

Yuting Ma; Heng Yang

doi:10.21769/BioProtoc.2076

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Peer-reviewed

In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death

采用体外分析法检测细胞死亡后的钙网蛋白暴露、ATP和HMGB1释放

Yuting Ma

HY Heng Yang

发布: 2016年12月20日第6卷第24期 DOI: 10.21769/BioProtoc.2076 浏览次数: 19066

评审: Lee-Hwa TaiJustine MarsolierMichael Enos

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Abstract

Accumulating evidence is revealing the essential role of immune system in cancer treatment. Certain chemotherapeutic drugs can potently induce the release of ‘cell death associated molecular patterns’ (CDAMPs), which accompanies cancer cell demise. CDAMPs can engage corresponding receptors on immune cells and stimulate immune responses to achieve long-term tumor control (Ma et al., 2013; Ma et al., 2014; Yang et al., 2015). Among reported CDAMPs, calreticulin (CALR), ATP and HMGB1 are well known for their immune-stimulatory effect. Here we describe the assays that we applied to measure cell death and these CDAMPs. Briefly, cell death can be analyzed by co-staining of 4’,6-diamidino-2-phenylindole (DAPI) with 3,3’-Dihexyloxacarbocyanine Iodide [DiOC6(3)] or Annexin V. CALR exposure on the cell membrane can be detected by flow cytometry. ATP and HMGB1 release can be quantified by luminescence assay and ELISA assay respectively.

Keywords: Cell death (细胞死亡)

Background

Lactate dehydrogenase assay and trypan blue staining are traditional methods to examine cell death. We describe here two feasible and economic solutions to detect apoptotic and necrotic cell death by flow cytometry (FCM). DAPI labels cells with disrupted integrity (necrosis), while Annexin V binds to phosphatidylserine (which is externalized upon apoptosis). DiOC6(3) uptake indicates mitochondrial transmembrane potential (MTP) and the collapse of MTP reveals apoptosis. DAPI doesn’t require compensation with phycoerythrin (PE, which is conjugated with Annexin V protein) or DiOC6(3), and therefore show advantage over propidium iodide (PI) in these assays.

CALR is an endoplasmic reticulum protein, and activation of caspase cascades triggers CALR translocation to cytoplasmic membrane. CALR exposure can be detected by immunofluorescent staining with corresponding antibodies, followed by FCM- or microscopy-based detection. Extracellular ATP can be measured by enzymatic activity of Luciferase, while HMGB1 concentration can be detected by sensitive ELISA kits.

Materials and Reagents

24 well plates
2 ml Eppendorf tubes
MCA205 fibrosarcoma cells (H2^b) were induced by 3-methylcholanthrene in C57BL/6 mice (Shu et al., 1985). The assays described here are applicable for other cell lines
DMEM high glucose (Thermo Fisher Scientific, Gibco^TM, catalog number: 11965092 , or GE Healthcare, HyClone^TM , catalog number: SH30022 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 16000044 )
100 mM sodium pyruvate solution (Thermo Fisher Scientific, Gibco^TM, catalog number: 11360070 )
HEPES, 1 M buffer solution (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630080 )
Penicillin and streptomycin
Mitoxantrone (Sigma-Aldrich, catalog number: M6545 )
Trypsin (0.25%) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15050065 )
1x PBS, pH 7.4 (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010031 )
DiOC6(3) (3,3’-dihexyloxacarbocyanine Iodide) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D273 )
DAPI (4’,6-diamidino-2-phenylindole, dihydrochloride) (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: D1306 )
CALR antibody (Ab): rabbit monoclonal Ab, clone number: EPR3924 (Abgent, catalog number: AJ1124a ; or Abcam, catalog number: ab92516 ); or rabbit polyclonal Ab (Abcam, catalog number: ab2907 )
Alexa488 conjugated goat anti-rabbit IgG (H+L) secondary antibody (Thermo Fisher Scientific, Invitrogen, catalog number: A-11008 )
ATP Bioluminescent Assay Kit (Sigma-Aldrich, catalog number: FL-AA ) or ENLITEN^® ATP Assay System (Promega, catalog number: FF2000 )
Annexin V Apoptosis Detection Kit (BD, catalog number: 559763 )
HMGB1 ELISA Kit (Tecan Trading, catalog number: ST51011 )
1x Annexin V binding buffer (see Recipes)
ATP assay mix (see Recipes)
rLuciferase/Luciferin buffer(see Recipes)

Equipment

Centrifuge
Water-Jacketed CO₂ incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: HERAcell^® 150i )
Multi-channel pipette
SpectraMax L microplate reader (Molecular Device, model: SpectraMax L )
SpectraMax i3 multi-mode detection platform (Molecular Device, model: SpectraMax i3 )
Attune^TM NxT flow cytometer (Thermo Fisher Scientific, model: Attune^TM NxT Flow Cytometer )

Note: Alternatively, VICTOR^TM X multilabel reader (PerkinElmer) and MACSQuant^® Analyzer 10 (Miltenyi Biotec) are also suitable.

Software

Excel (Microsoft Office)
GraphPad Prism (San Diego, CA, USA)
FlowJo software (Treestar Inc., Ashland, OR, USA)

Procedure

English

中文翻译

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如何引用

Ma, Y. and Yang, H. (2016). In vitro Assays for the Detection of Calreticulin Exposure, ATP and HMGB1 Release upon Cell Death. Bio-protocol 6(24): e2076. DOI: 10.21769/BioProtoc.2076.