In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica
痢疾变形虫的体外荧光基质降解试验
发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1769 浏览次数: 8437
评审: Fanglian HeAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Feb 2015
Abstract
Fluorescent matrix degradation assay is a popular and widely used assay in the field of invadopodium biology (Artym et al., 2009). Matrix remodeling and degradation can be observed under both physiological and pathological conditions. Cancer cells extensively remodel and degrade the underlying matrix by employing actin-rich protrusive structures called invadosomes. Similar structures are formed by the protozoan parasite Entamoeba histolytica (E. histolytica), upon coming in contact with fibronectin, a major component of the host (extracellular matrix) ECM. Here, we describe a similar assay to measure matrix degradation by Entamoeba histolytica.
Materials and Reagents
- Sterile disposable pipettes (10 ml and 5 ml)
- Aluminum foil
- Four well tissue culture plates (Thermo Fisher Scientific, NuncTM, catalog number: 176740 )
- Round Glass coverslips (12 mm) (Bellco Glass, catalog number: 1943-10012A )
- Parafilm
- Entamoeba histolytica HM1:IMSS strain (a kind gift from Prof. Alok Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India)
- Fluorescently labeled fibronectin (CYTOSKELETON, catalog number: FNR02 )
- Glutaraldehyde (25% stock solution in dH2O, EM grade) (Sigma-Aldrich, catalog number: G5882 )
- Sodium Borohydride (NaBH4) (Sigma-Aldrich, catalog number: 452882 )
- Poly-L-lysine (Sigma-Aldrich, catalog number: P8920 )
- 70% ethanol (Merck Millipore Corporation)
- Alexa 568 Phalloidin (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A12380 )
- 1% BSA (Sigma-Aldrich, catalog number: A-7906 ) in PBS
- Mowiol mounting medium (Sigma-Aldrich, catalog number: 81381 )
- Complete BI medium (incomplete supplemented with 15%, Adult Bovine Serum)
- GasPakTM EZ Anaerobe Pouch System (BD Biosciences, catalog number: 260683 )
- Sterile dH2O
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- KCl
- Potassium phosphate monobasic (Na2HPO4) (Sigma-Aldrich, catalog number: P5655 )
- Potassium phosphate dibasic trihydrate (KH2PO4) (Sigma-Aldrich, catalog number: P5504 )
- NaOH
- Hilyte 488 Fibronectin
- Biosate peptone BBL (BD Biosciences, catalog number: 211862 )
- Glucose (Dextrose) (Sigma-Aldrich, catalog number: D9434 )
- L-cysteine (Sigma-Aldrich, catalog number: C1276 )
- L-ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
- Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
- 10x phosphate-buffered saline (PBS) (see Recipes)
- 0.01% poly-L-lysine solution (see Recipes)
- 0.5% glutaraldehyde solution (see Recipes)
- 5 mg/ml sodium borohydride (see Recipes)
- 4% paraformaldehyde (PFA) (see Recipes)
- 1% BSA in PBS (see Recipes)
- 0.1% Triton-X-100 (see Recipes)
- 100 μg/ml Hilyte488 Fibronectin (FNR02) (see Recipes)
- Basal incomplete medium (see Recipes)
- Complete medium (see Recipes)
Equipment
- Laminar air flow hood
- 37 °C incubator
- Forceps
- Moist chamber
- Vacuum Pump and glass Pasteur pipettes with tubings
- Table top centrifuge with swinging bucket rotor
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Emmanuel, M. and Datta, S. (2016). In vitro Fluorescent Matrix Degradation Assay for Entamoeba histolytica. Bio-protocol 6(6): e1769. DOI: 10.21769/BioProtoc.1769.
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分类
微生物学 > 微生物-宿主相互作用 > 体内实验模型
细胞生物学 > 细胞成像 > 荧光
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