肿瘤物质摄取的量化

Richard N. Hanna; Catherine C. Hedrick

doi:10.21769/BioProtoc.1974

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
登录
/
注册
- 个人主页
- 编辑个人信息
- 修改密码
- 退出
CN
- EN - English
- CN - 中文

Peer-reviewed

Quantification of Tumor Material Uptake

肿瘤物质摄取的量化

Richard N. Hanna email

CH Catherine C. Hedrick

发布: 2016年10月20日第6卷第20期 DOI: 10.21769/BioProtoc.1974 浏览次数: 9917

评审: Kristopher MarjonMartin V KolevElizabeth V. Clarke

PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Science, featuring study using the protocol.

Nov 2015

Submit your protocol to the Special Issue on Technologies for RNA Detection

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

利用小鼠脾细胞分离并离体检测CD8⁺ T细胞的分裂与激活

Melissa Dolan [...] John M.L. Ebos

2025年08月20日 3487 阅读

利用 Guava<sup>®</sup> 自噬检测试剂盒和流式细胞术检测人外周血单个核细胞的自噬

利用 Guava^® 自噬检测试剂盒和流式细胞术检测人外周血单个核细胞的自噬

Melanie Scherer [...] Jörg Bergemann

2025年09月20日 1340 阅读

外周血中细胞外囊泡的分离与分析方法：红细胞、内皮细胞及血小板来源的细胞外囊泡

Bhawani Yasassri Alvitigala [...] Lallindra Viranjan Gooneratne

2025年11月05日 1328 阅读

Abstract

Extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris may help cancers invade new organs. Enhancing the removal of extracellular tumor material by immune cells represents a novel immunotherapy approach for preventing cancer metastasis. This protocol quantifies the uptake and removal of extracellular tumor material from circulation and tissues by immune cells. In this assay fluorescent tumor cells are transferred into mice, and then immune cells are quantified by either flow cytometry or imaging cytometry for their uptake of tumor material.

Keywords: Cancer (癌症)

Background

Recent studies have demonstrated that extracellular tumor material including exosomes, microvesicles and apoptotic tumor debris shed from tumors are important mediators of tumor metastasis, growth and evasion of the immune response (Vader et al., 2014; Pucci and Pittet, 2013; Pucci et al., 2016). Immune cells have the ability to remove, respond and transport this circulating tumor material (Hanna et al., 2015; Pucci et al., 2016; Headley et al., 2016). This protocol offers a novel approach to quantify tumor material uptake by specific immune cell populations, and may be adapted to test immune targets that regulate tumor material uptake. This protocol may assist in better understanding the immune response to extracellular tumor material, with the hope of eventually developing novel therapies targeting extracellular tumor material in cancer development.

Materials and Reagents

30 gauge insulin syringe (BD, Ultra-Fine^TM, catalog number: 328431 )
15 ml tubes
70 μm cell strainers (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22363548 )
15 mm Petri dish
5 ml syringe (BD, Luer-Lok^TM, catalog number: 309646 )
96-well v-bottom plate
Lewis lung carcinoma cells expressing red fluorescent protein (LLC-RFP), B16F10 green fluorescent protein (B16F10-GFP) or other fluorescent-tagged tumor cell line (AntiCancer.com, http://www.anticancer.com/Fluorescent_protein_cell_lines_April_2010.pdf)
Syngeneic recipient mice. C57BL/6J mice (The Jackson Laboratory, catalog number: 000664 ) for LLC and B16F10 tumors.
TrypLE (Thermo Fisher Scientific, Gibco^TM, catalog number: 12604013 )
Dulbecco's phosphate-buffered saline (DPBS) (GE Healthcare, Hyclone^TM, catalog number: SH30013.02 )
UltraPure^TM 0.5 M EDTA, pH 8.0 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15575020 )
Red blood cell (RBC) lysis buffer (10x) (BioLegend, catalog number: 420301 )
Fc Block (CD16/32) (BD, Pharmingen^TM, catalog number: 553141 )
Fluorochrome conjugated antibodies:
CD11b-FITC (BioLegend, catalog number: 101206 )
CD115-APC (BioLegend, catalog number: 135510 )
Ly6C-APC-Cy7 (BioLegend, catalog number: 128026 )
CD45-BV421 (BioLegend, catalog number: 103134 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9418 )
Live/Dead Fixable Blue (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: L34962 )
Sodium azide (Sigma-Aldrich, catalog number: S2002 )
Flow buffer (FB) (see Recipes)
RBC lysis buffer (see Recipes)

Equipment

Cell culture flask
Centrifuge
Multiparameter flow cytometer with optimal excitation and detectors for tumor fluorophore (BD, LSRII or similar) or Amnis imaging cytometer (ImageStream X Mark II)

Software