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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   

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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

  1. Sterile disposable pipettes (10 ml and 5 ml)
  2. Aluminum foil
  3. Four well tissue culture plates (Thermo Fisher Scientific, NuncTM, catalog number: 176740 )
  4. Round Glass coverslips (12 mm) (Bellco Glass, catalog number: 1943-10012A )
  5. Parafilm
  6. Entamoeba histolytica HM1:IMSS strain (a kind gift from Prof. Alok Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India)
  7. Fluorescently labeled fibronectin (CYTOSKELETON, catalog number: FNR02 )
  8. Glutaraldehyde (25% stock solution in dH2O, EM grade) (Sigma-Aldrich, catalog number: G5882 )
  9. Sodium Borohydride (NaBH4) (Sigma-Aldrich, catalog number: 452882 )
  10. Poly-L-lysine (Sigma-Aldrich, catalog number: P8920 )
  11. 70% ethanol (Merck Millipore Corporation)
  12. Alexa 568 Phalloidin (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A12380 )
  13. 1% BSA (Sigma-Aldrich, catalog number: A-7906 ) in PBS
  14. Mowiol mounting medium (Sigma-Aldrich, catalog number: 81381 )
  15. Complete BI medium (incomplete supplemented with 15%, Adult Bovine Serum)
  16. GasPakTM EZ Anaerobe Pouch System (BD Biosciences, catalog number: 260683 )
  17. Sterile dH2O
  18. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  19. KCl
  20. Potassium phosphate monobasic (Na2HPO4) (Sigma-Aldrich, catalog number: P5655 )
  21. Potassium phosphate dibasic trihydrate (KH2PO4) (Sigma-Aldrich, catalog number: P5504 )
  22. NaOH
  23. Hilyte 488 Fibronectin
  24. Biosate peptone BBL (BD Biosciences, catalog number: 211862 )
  25. Glucose (Dextrose) (Sigma-Aldrich, catalog number: D9434 )
  26. L-cysteine (Sigma-Aldrich, catalog number: C1276 )
  27. L-ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
  28. Ferric ammonium citrate (Sigma-Aldrich, catalog number: F5879 )
  29. 10x phosphate-buffered saline (PBS) (see Recipes)
  30. 0.01% poly-L-lysine solution (see Recipes)
  31. 0.5% glutaraldehyde solution (see Recipes)
  32. 5 mg/ml sodium borohydride (see Recipes)
  33. 4% paraformaldehyde (PFA) (see Recipes)
  34. 1% BSA in PBS (see Recipes)
  35. 0.1% Triton-X-100 (see Recipes)
  36. 100 μg/ml Hilyte488 Fibronectin (FNR02) (see Recipes)
  37. Basal incomplete medium (see Recipes)
  38. Complete medium (see Recipes)

Equipment

  1. Laminar air flow hood
  2. 37 °C incubator
  3. Forceps
  4. Moist chamber
  5. Vacuum Pump and glass Pasteur pipettes with tubings
  6. Table top centrifuge with swinging bucket rotor

Procedure

Video 1. In vitro fluorescent matrix degradation assay for Entamoeba histolytica

Coating of glass coverslips with fluorescent matrix

  1. Clean the glass coverslips with acetone, air dry and sterilize them by autoclaving before use.
  2. Further, handle the coverslips under sterile conditions inside the laminar air flow hood. Transfer the coverslips to a four well tissue culture plate using ethanol wiped forceps.
  3. Coat each coverslip with 0.01% poly-L-lysine using 80 μl of poly-L-lysine solution per coverslip for 20 min at room temperature. Air dry the solution under the hood.
  4. Wash the coverslips by adding 500 μl of sterile 1x PBS gently to the side of the well and incubating for 5 min each. The poly-L-lysine coated side should face up during the washes. After each wash aspirate it gently using a sterile glass pasteur pipette attached to a vacuum pump and repeat the washes for three times.
  5. After the third PBS wash, add 500 μl of ice cold 0.5% glutaraldehyde to each well such that the coverslips are immersed and incubate under dark at room temperature for 15 min.
  6. Aspirate the glutaraldehyde solution gently by tilting the plate to an angle such that coverslips are not disturbed and again wash with sterile 1x PBS. Repeat the washes for three times.
  7. Make a humid chamber using tissue paper and 70% ethanol. Neatly make a stack of tissue paper by folding it over and moisten it by spraying with 70% ethanol. Place the stack in a sterile Petri dish to make a humid chamber. Place a neatly cut parafilm over the moist tissue paper stack and press it gently so that it sticks well.
  8. Now, place appropriate number of drops of 50 μl of prewarm (37 °C, preferably in a water bath) fluorescently labeled fibronectin (100 μg/ml) over the parafilm according to the number of coverslips being coated.
  9. Carefully lift the coverslips from the wells holding at the edges using a pair of forceps and place them with their poly-L-lysine coated side facing downwards on the drop of the fluorescent fibronectin on the parafilm. While placing the coverslips, ensure that there is no air bubble trapped beneath them. If so, gently press the coverslips with the forceps to eliminate the trapped air bubbles.
  10. Incubate under safe light in a 37 °C incubator for an hour.
  11. After an hour, remove the coverslips from the incubator. Now coverslips should be handled in safe light. Lift the coverslips carefully one-by-one with the help of the forceps without scratching the matrix and place them with their coated side facing up in a fresh tissue culture plate.
  12. Again wash them gently with 1x PBS as done previously in step 4. Meanwhile, prepare fresh 5 mg/ml solution of sodium borohydride by dissolving it in 1x PBS. Add 500 μl of sodium borohydride per well such that the coverslips are immersed in it. Incubate under dark at room temperature for 15 min. As sodium borohydride bubbles upon dissolution, make sure that the bubbles do not settle on the coverslips by gently rocking them back and forth.
  13. Wash the coverslips with 1x PBS for three times and then finally sterilize by soaking them into 70% ethanol for 10 min at room temperature. Further wash the coverslips with sterile 1x PBS and then finally add 500 μl of complete BI medium to each well and incubate at 37 °C in an incubator for an hour.
  14. Coverslips are now ready to be used. If you do not want to use them immediately, sterilize them using 70% ethanol and then store them at 4 °C under dark for up to a week.

Sample preparation for the assay

  1. Take a confluent tube of Entamoeba histolytica HM1: IMSS strain in logarithmic phase and remove the used medium. Add 10 ml of fresh complete BI medium and transfer the glass tube on ice for 5 min so as to detach the cells. The culture volume in the glass tube is 13 ml.
  2. Now gently tap the tube to completely detach the trophozoites and transfer in a 15 ml conical tube. Take a small aliquot to count the number of cells using haemocytometer. Spin them down at 500 x g for 5 min at room temperature. Now resuspend the cell pellet in warm complete BI medium to a concentration of 40 x 104 cells/ml.
  3. Add 0.5 ml of cells i.e. 2 x 105 cells per well over the fluorescently matrix coated coverslips and place the tissue culture plate inside a GasPak EZ pouch to maintain anaerobic conditions. Place the whole setup in 37 °C incubator.
  4. Incubate for 36-48 h at 37 °C under dark.
  5. Close to the end of incubation, warm an aliquot of 1x PBS and 4% PFA at 37 °C for further use.

Preparing samples for fluorescence microscopy

  1. Aspirate the medium by tilting the plate to an angle without disturbing the coverslips. Now wash the cells with warm sterile 1x PBS and fix the cells immediately using 4% PFA. Incubate at room temperature for 15 min under dark.
  2. After fixation, wash the sample with 1x PBS and permeabilize with 0.1% Triton-X-100 at room temperature for 10 min. Wash again and block the samples with 1% BSA in PBS for an hour at room temperature.
  3. To label with Alexa 568 Phalloidin, dilute phalloidin in a ratio of 1:100 in the blocking solution (1% BSA in PBS).
  4. Transfer the coverslips carefully to a humid chamber again and add 50 μl of Alexa 568 Phalloidin to each coverslip and incubate under dark at room temperature for an hour.
  5. Subsequently, wash the samples by again placing them back in the tissue culture plate and adding 1x PBS.
  6. After the final wash, mount the sample using mounting medium. Let the samples air dry at room temperature under dark.
  7. Examine the fluorescence of the matrix and the samples using epifluorescence or confocal microscope with appropriate light source and filter sets. Since the matrix layer formed is very thin the samples should be focused accurately so that the entire field of view has uniform fluorescence. The degradation of the matrix can be observed as loss of fluorescence around the cells and would appear as a dark halo surrounding the cells.


    Figure 1. Rab21 mediated fibronection degradation by amoebic trophozoites. A. Trophozoites overexpressing Rab21CA mutant were plated on a Hilyte 488 fibronectin coated glass coverslip and incubated for 48 h at 37 °C under dark. Cells were fixed, permeabilized and stained with Alexa 568 Phalloidin and DAPI. B. Undamaged Hilyte 488 Fibronectin. Scale bar, 10 µm.

Notes

  1. Always prepare fresh 0.5% glutaraldehyde solution. As this solution is hazardous, avoid contact with skin and eyes.
  2. Sodium borohydride should also be prepared fresh just before use. Upon dissolution, sodium borohydride releases free H2 gas, so it should always be prepared in conical tubes with enough free space to avoid spills and bumping of the solution. Do not tightly cap the tubes containing the solution.
  3. Passage your cells regularly and do not allow cells to grow to 100% confluency. Use cells which are 70-80% confluent for the experiment.

Recipes

  1. 10x phosphate-buffered saline (PBS)
    Dissolve 80 g NaCl with 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4
    Adjust pH to 7.4 with NaOH
    Add dH2O to 1,000 ml
    Filter sterilize (0.2 μm)
    Stored at room temperature
  2. 0.01% poly-L-lysine solution
    Dilute 0.1% solution of poly-L-lysine in sterile dH2O in a 1:10 ratio
  3. 0.5% glutaraldehyde solution
    Add 0.2 ml of stock solution to 10 ml of sterile 1x PBS
  4. 5 mg/ml sodium borohydride
    Weigh 100 mg of sodium borohydride
    Dissolve in 20 ml of 1x PBS just before use
  5. 4% PFA
    Weigh 4 g of PFA
    Dissolve in 100 ml of pre-warmed 1x PBS with constant stirring
  6. 1% BSA in PBS
    Weigh 0.5 g of BSA and dissolve in 50 ml of 1x PBS
  7. 0.1% Triton-X-100
    Make a stock solution of 10% Triton-X-100 in 1x PBS
    Add 0.5 ml of 10% Triton-X-100 in 50 ml of 1x PBS
  8. 100 μg/ml Hilyte488 Fibronectin (FNR02)
    Hilyte 488 Fibronectin comes as a desiccated powder and is stored at 4 °C under dark Dissolve the powder at a final concentration of 1 mg/ml using sterile dH2O
    Make aliquots and stored at -20 °C
  9. Basal incomplete medium
    Dissolve:
    3 g of Biosate peptone BBL
    1 g of Glucose
    200 mg of NaCl
    60 mg of KH2PO4
    100 mg of K2HPO4
    100 mg of L-cysteine
    20 mg of L-ascorbic acid and 2.28 mg of Ferric ammonium citrate in dH2O
    Adjust the pH to 6.8 using 8 N NaOH
    Add dH2O to 88 ml
    Sterilized by autoclaving at 121 °C at 15 psi for 15 min
  10. Complete medium
    Add 15 ml of adult bovine serum (filter sterilized) and 2 ml of 100x vitamin mixture (filter sterilized) to 88 ml of basal incomplete medium

Acknowledgments

We would like to thank Prof. Alok Bhattacharya (Jawaharlal Nehru University, New Delhi, India) and Dr. Sandipan Ganguly (National Institute of Cholera and Enteric Diseases, Kolkatta, India) for providing us with the Entamoeba histolytica HM1: IMSS strain. We are grateful to Professor Stefan Linder (University of Hamburg, Hamburg, Germany) for helpful discussion and suggestions. We are also thankful to Drs. Vira V. Artym, Kenneth M. Yamada and Susette C. Mueller as the protocol for E. histolytica has been adapted from their published protocol for studying cancer cell invasion in Methods in Molecular Biology. We sincerely thank Ms. Lekha Vinod Shah, final year Dual BS-MS degree program student at IISER Bhopal, Department of Biological Sciences, who shot and edited the video for the protocol.We are thankful to the Central Instrumentation Facility at IISER Bhopal, India. This work was funded by intramural funds from IISERB and funds from Max Planck Gesellschaft (MPG), Germany and Department of Science and Technology (DST), India. ME was funded by Senior Research Fellowship provided by University Grant Commission, Government of India.

References

  1. Artym, V. V., Yamada, K. M. and Mueller, S. C. (2009). ECM degradation assays for analyzing local cell invasion. Methods Mol Biol 522: 211-219.
  2. Emmanuel, M., Nakano, Y. S., Nozaki, T. and Datta, S. (2015). Small GTPase Rab21 mediates fibronectin induced actin reorganization in Entamoeba histolytica: implications in pathogen invasion. PLoS Pathog 11(3): e1004666.

简介

荧光基质降解测定是在invadopodium生物学领域中广泛使用的测定(Artym等人,2009)。 可以在生理和病理条件下观察基质重塑和降解。 癌细胞通过采用称为invadosomes的富含肌动蛋白的突出结构广泛地重塑和降解基础矩阵。 在与纤维连接蛋白(宿主(细胞外基质)ECM的主要组分)接触时,由原生动物寄生虫(即溶组织内阿米巴)( histolytica )形成类似的结构。 在这里,我们描述了通过溶组织内阿米巴测量基质降解的类似测定法。

材料和试剂

  1. 无菌一次性移液管(10ml和5ml)
  2. 铝箔
  3. 四孔组织培养板(Thermo Fisher Scientific,Nunc TM,目录号:176740)
  4. 圆形玻璃盖玻片(12mm)(Bellco Glass,目录号:1943-10012A)
  5. parafilm
  6. HM1:IMSS毒株(来自印度新德里Jawaharlal Nehru大学生命科学学院Alok Bhattacharya教授的赠品)
  7. 荧光标记的纤连蛋白(CYTOSKELETON,目录号:FNR02)
  8. 戊二醛(dH 2 O中25%储备液,EM级)(Sigma-Aldrich,目录号:G5882)
  9. 硼氢化钠(NaBH 4)(Sigma-Aldrich,目录号:452882)
  10. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P8920)
  11. 70%乙醇(Merck Millipore Corporation)
  12. Alexa 568鬼笔环肽(Thermo Fisher Scientific,Molecular Probes ,目录号:A12380)
  13. 1%BSA(Sigma-Aldrich,目录号:A-7906)的PBS
  14. Mowiol封固剂(Sigma-Aldrich,目录号:81381)
  15. 完全BI培养基(不完全补充15%,成人牛血清)
  16. GasPak TM EZ Anaerobe Pouch System(BD Biosciences,目录号:260683)
  17. 无菌dH 2 O 2/b
  18. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  19. KCl
  20. 磷酸二氢钾(Na 2 HPO 4)(Sigma-Aldrich,目录号:P5655)
  21. 磷酸氢二钾三水合物(KH 2 PO 4)(Sigma-Aldrich,目录号:P5504)
  22. NaOH
  23. Hilyte 488纤连蛋白
  24. 生物蛋白胨BBL(BD Biosciences,目录号:211862)
  25. 葡萄糖(葡萄糖)(Sigma-Aldrich,目录号:D9434)
  26. L-半胱氨酸(Sigma-Aldrich,目录号:C1276)
  27. L-抗坏血酸(Sigma-Aldrich,目录号:A5960)
  28. 柠檬酸铁铵(Sigma-Aldrich,目录号:F5879)
  29. 10x磷酸盐缓冲盐水(PBS)(见配方)
  30. 0.01%聚-L-赖氨酸溶液(见配方)
  31. 0.5%戊二醛溶液(见配方)
  32. 5mg/ml硼氢化钠(参见配方)
  33. 4%多聚甲醛(PFA)(参见配方)
  34. 1%BSA的PBS溶液(见配方)
  35. 0.1%Triton-X-100(参见配方)
  36. 100μg/ml Hilyte488纤连蛋白(FNR02)(见Recipes)
  37. 基本不完全培养基(见配方)
  38. 完整介质(见配方)

设备

  1. 层流气流罩
  2. 37℃孵育器
  3. 镊子
  4. 湿室
  5. 真空泵和带管道的玻璃巴斯德移液器
  6. 台式离心机,带有旋转叶片转子

程序

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视频1. 体外荧光基质降解试验阿米巴溶组织
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用荧光基质涂覆玻璃盖玻片

  1. 用丙酮清洁玻璃盖玻片,风干,使用前通过高压灭菌消毒。
  2. 此外,在无菌条件下处理盖玻片 层流气流罩。将盖玻片转移到四井组织 培养板使用乙醇擦拭镊子
  3. 每个盖玻片 用0.01%聚-L-赖氨酸,使用80μl聚-L-赖氨酸溶液 盖玻片在室温下20分钟。空气干燥下的溶液 ?罩
  4. 通过加入500μl的无菌1x PBS洗涤盖玻片 轻轻地到孔的侧面并孵育5分钟。的 聚-L-赖氨酸涂层的侧面应当在洗涤期间面向上。之后每个 使用无菌玻璃巴斯德吸管轻轻吸取它 到真空泵并重复洗涤三次
  5. 之后 ?第三次PBS洗涤,向每孔中加入500μl冰冷的0.5%戊二醛 ?使得盖玻片浸没并在室温下在黑暗下孵育 温度15分钟
  6. 吸出戊二醛溶液 轻轻地倾斜板的角度,使盖玻片不 干扰并再次用无菌1x PBS洗涤。重复洗涤 三次。
  7. 使用薄纸和70% 乙醇。整齐地做一叠纸巾通过折叠和 通过喷洒70%乙醇来润湿。将堆放在无菌的 petridish做一个潮湿的房间。把一个整齐切割的石蜡膜放在 湿纸巾叠,轻轻按压,使其粘贴良好
  8. 现在,放置适当数量的50微升预热(37°C, 优选在水浴中)荧光标记的纤连蛋白(100μg/ml) 根据涂布的盖玻片的数量在石蜡膜上
  9. 小心地从保持在边缘的井提起盖玻片 使用一对镊子并将它们与聚-L-赖氨酸涂层 侧面向下在荧光纤连蛋白的滴上 石蜡膜。放置盖玻片时,确保没有空气 气泡陷在他们下面。如果是这样,轻轻地按盖玻片 ?镊子,以消除被困的气泡
  10. 在37°C孵育箱中在安全光照下孵育1小时
  11. 一小时后,从培养箱中取出盖玻片。现在 盖玻片应在安全光线下处理。提起盖玻片 仔细一个一个地用镊子的帮助不刮伤 ?基质,并将它们的涂层面朝上放在新鲜 组织培养板
  12. 再次用1x PBS轻轻地洗它们 ?同时,制备新鲜的5mg/ml的溶液 硼氢化钠溶解在1×PBS中。加入500μl钠 硼氢化物,使得盖玻片浸入其中。 在黑暗中室温孵育15分钟。作为钠 硼氢化物气泡在溶解时确保气泡不会 通过轻轻地来回摇摆来安置在盖玻片上
  13. 用1x PBS洗涤盖玻片三次,然后终于 通过将其在室温下浸泡在70%乙醇中10分钟来灭菌 温度。进一步用无菌1×PBS洗涤盖玻片,然后 最后向每个孔中加入500μl完全BI培养基并在37℃下孵育 ?℃的培养箱中培养1小时
  14. 盖玻片现在准备好了 用过的。如果你不想使用它们立即消毒他们使用 70%乙醇,然后将其在4℃下在黑暗中储存长达一周。

  1. 取合并的溶组织内阿米巴HM1:IMSS菌株 对数相并除去使用的介质。加入10毫升新鲜 完全BI培养基,并将玻璃管在冰上转移5分钟 ?分离细胞。玻璃管中的培养体积为13ml
  2. 现在轻轻点击管,以完全分离滋养体和 转移到15ml锥形管中。取一小部分计数 使用血细胞计数器。将它们以500 x g 旋转5秒 min。现在重新悬浮细胞沉淀在温暖的完成 BI培养基中至浓度为40×10 4个细胞/ml
  3. 加入0.5ml 细胞,即每孔超过荧光基质包被的2×10 5个细胞 盖玻片,并将组织培养板放置在GasPak EZ袋内 以维持厌氧条件。将整个设置在37°C 孵化器。
  4. 在37℃,黑暗下孵育36-48小时
  5. 在接近培养结束时,在37℃下加热1×PBS和4%PFA的等分试样以备进一步使用。

准备样品用于荧光显微镜

  1. 通过将板倾斜到一个角度而不干扰,吸出介质 盖玻片。现在用温热的无菌1x PBS洗涤细胞,并修复 细胞立即使用4%PFA。在室温下孵育15分钟 暗处。
  2. 固定后,用1x PBS洗涤样品 用0.1%Triton-X-100在室温下透化10分钟。洗 ?再次在室温下用含有1%BSA的PBS封闭样品1小时 温度
  3. 为了用Alexa 568鬼笔环素标记,在封闭溶液(PBS中的1%BSA)中以1:100的比例稀释鬼笔环肽。
  4. 将盖玻片小心地转移到潮湿的房间,并再次添加 50μl的Alexa 568鬼笔环肽到每个盖玻片,并在黑暗中孵育 在室温下搅拌1小时
  5. 随后,通过将样品再次放回组织培养板并加入1×PBS来洗涤样品
  6. 最后一次洗涤后,使用固定介质安装样品。让样品在室温下在黑暗下风干
  7. 使用检查基质和样品的荧光 落射荧光或共聚焦显微镜与适当的光源和 ?过滤器集。因为所形成的基质层非常薄 应该准确地聚焦,以便整个视场都有 均匀荧光。可以观察到基质的降解 细胞周围的荧光丧失,并且将表现为暗晕 围绕细胞。


    图1。 A.滋养体过表达 Rab21CA突变体涂布在Hilyte 488纤连蛋白包被的玻璃上 盖玻片并在37℃在黑暗下孵育48小时。细胞被固定, 渗透并用Alexa 568鬼笔环肽和DAPI染色。 B.未受损的Hilyte 488纤连蛋白。比例尺, ?10μm

笔记

  1. 总是准备新鲜的0.5%戊二醛溶液。由于此溶液有危险,请避免接触皮肤和眼睛。
  2. 硼氢化钠也应该在使用前准备好。溶解后,硼氢化钠释放游离的H 2气体,因此其应当总是在具有足够自由空间的锥形管中制备以避免溶液的溢出和碰撞。不要紧紧盖住装有溶液的试管
  3. 定期传代细胞,不允许细胞生长至100%融合。使用70-80%汇合的细胞用于实验

食谱

  1. 10x磷酸盐缓冲盐水(PBS)
    将80g NaCl溶于2g KCl,14.4g Na 2 HPO 4,2.4g KH 2 PO 4溶液,
    用NaOH调节pH至7.4 将dH <2> O添加至1,000 ml
    过滤灭菌(0.2μm)
    在室温下贮存
  2. 0.01%聚-L-赖氨酸溶液 在无菌的dH 2 O中以1:10的比例稀释0.1%的聚-L-赖氨酸溶液
  3. 0.5%戊二醛溶液
    将0.2ml储备溶液加入10ml无菌1x PBS中
  4. 5mg/ml硼氢化钠
    称量100mg硼氢化钠
    在使用前溶于20 ml 1×PBS中
  5. 4%PFA
    称量4g PFA
    在恒定搅拌下溶于100ml预热的1×PBS中
  6. 1%BSA的PBS溶液中 称取0.5g BSA,并溶解于50ml 1x PBS中
  7. 0.1%Triton-X-100 制备10%Triton-X-100在1x PBS中的储备溶液
    加入0.5ml的10%Triton-X-100在50ml的1x PBS中
  8. 100μg/ml Hilyte488纤连蛋白(FNR02) Hilyte 488纤连蛋白作为干燥粉末,并在4℃下在黑暗下储存。使用无菌dH 2 O 2/d溶解粉末至终浓度为1mg/ml。 将等分试样并保存在-20°C
  9. 基本不完全培养基
    溶解:
    3克生物蛋白胨BBL 1g葡萄糖
    200 mg NaCl
    60mg的KH 2 PO 4 sub/
    100mg的K 2 HPO 4
    100mg L-半胱氨酸 20mg L-抗坏血酸和2.28mg在dH 2 O中的柠檬酸铁铵
    用8N NaOH将pH调节至6.8 将dH <2> O添加至88ml
    在121℃,15psi下高压灭菌15分钟
  10. 完成媒介
    将15ml成年牛血清(过滤灭菌)和2ml 100×维生素混合物(过滤除菌)加入88ml基础不完全培养基中

致谢

我们要感谢Alok Bhattacharya教授(印度新德里的Jawaharlal Nehru大学)和Sandipan Ganguly博士(印度加尔各答的霍乱和肠道疾病国家研究所),为我们提供了溶组织内阿米巴> HM1:IMSS菌株。我们感谢Stefan Linder教授(德国汉堡大学)提供有益的讨论和建议。我们也感谢博士。 Vira V.Artym,Kenneth M.Yamada和Susette C.Mueller作为( histolytica )的方案已经改编自他们在Methods in Molecular Biology中研究癌细胞侵袭的已公布的方案。我们真诚地感谢Lekha Vinod Shah女士,最后一年是生物科学部IIShop博帕尔的双重BS-MS学位课程学生,他负责拍摄和编辑协议的视频。我们感谢印度的IISER Bhopal的中央仪器设施。这项工作由来自IISERB的内部资金和德国马克斯普朗克协会(MPG)和印度科学技术部(DST)的资金资助。 ME由印度政府大学奖学金委员会提供的高级研究奖学金资助。

参考文献

  1. Artym,V.V.,Yamada,K.M.and Mueller,S.C。(2009)。 用于分析局部细胞侵袭的ECM降解测定法 em。522:211-219。
  2. Emmanuel,M.,Nakano,Y. S.,Nozaki,T。和Datta,S。(2015)。 小GTP酶Rab21介导纤维连接蛋白诱导的肌动蛋白重组在溶组织内阿米巴中::病原体侵袭。 PLoS Pathog 11(3):e1004666。
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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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