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Chitinases are chitin-degrading enzymes. Chitinases play essential roles in combating chitin-containing pathogens as well as established roles in asthmatic inflammation. This assay is designed to detect chitinase activity in macrophage cell lysates. The chitin substrate is labeled with 4-methylumbelliferone. Hydrolysis of chitin releases 4-methylumbelliferone, and is measured fluorometrically to determine chitinase activity.

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Chitinase Assay from Cultured Bone Marrow Derived Macrophages
体外培养骨髓源巨噬细胞的几丁质酶测定

免疫学 > 免疫细胞功能 > 巨噬细胞
作者: Danielle Worth
Danielle WorthAffiliation: Division of Biomedical Sciences, University of California, Riverside, USA
Bio-protocol author page: a1031
J. Philip Nance
J. Philip NanceAffiliation: Division of Biomedical Sciences, University of California, Riverside, USA
Bio-protocol author page: a1032
 and Emma H. Wilson
Emma H. WilsonAffiliation: Division of Biomedical Sciences, University of California, Riverside, USA
For correspondence: emmaw@ucr.edu
Bio-protocol author page: a1033
Vol 3, Iss 23, 12/5/2013, 2461 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.983

[Abstract] Chitinases are chitin-degrading enzymes. Chitinases play essential roles in combating chitin-containing pathogens as well as established roles in asthmatic inflammation. This assay is designed to detect chitinase activity in macrophage cell lysates. The chitin substrate is labeled with 4-methylumbelliferone. Hydrolysis of chitin releases 4-methylumbelliferone, and is measured fluorometrically to determine chitinase activity.

[Abstract]

Materials and Reagents

  1. Cells to be analyzed. e.g. Bone Marrow-derived macrophages from C57BL/6 mice
  2. L-cell
  3. cDMEM/F12
  4. 4-Mehtylumbelliferyl labeled substrates:
    1. 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (exochitinase activity; β–N-acetylglucosaminidase activity) (Sigma-Aldrich, catalog number: M2133 )
    2. 4-Methylumbelliferyl N,N′-diacetyl-β-D-chitobioside (exochitinase activity; chitobiosidase activity) (Sigma-Aldrich, catalog number: M9763 )
    3. 4-Methylumbelliferyl β-D-N,N′,N′′-triacetylchitotriose (endochitinase activity) (Sigma-Aldrich, catalog number: M5639 )
  5. Chitinase from Trichoderma viride (Sigma-Aldrich, catalog number: C6242 )
  6. Methylumbelliferone Standard (50 mg/ml) (Sigma-Aldrich, catalog number: M1381 )
  7. PBS
  8. DMSO
  9. Plate reader
  10. Diabasic Sodium Phosphate
  11. Citric Acid
  12. Glycine
  13. DTT
  14. Assay buffer (see Recipes)
  15. Stop buffer (see Recipes)
  16. Protein lysis buffer (see Recipes)
  17. Substrate Stock (see Recipes)
  18. Positive control (see Recipes)

Equipment

  1. Flat bottom black 96-well plates
  2. Fluorescent plate reader (Molecular Devices)

Procedure

  1. Cells to be analyzed.  We have used Bone Marrow-derived macrophages from C57BL/6 mice.
    1. In brief, bone marrow is flushed from tibias and femurs of 6-8 week old mice with 10 ml complete DMEM/F12 supplemented with 20% L-cell supernatant (day 0).  
    2. Bone marrow cells are plated in non tissue culture treated 10 cm petri dishes ~10 ml cells per dish.  After 4 days, add 10 ml additional media.  
    3. Cells are harvested by gentle scraping on day 7.  
    4. Macrophages are plated at 50,000 cells/well in 96 well plates in cDMEM/F12 in 10% L-cell supernatant and allowed to rest for 3 days.
    5. On day 10, media is changed to cDMEM/F12 without L-cell supernatant and rested overnight.  
    6. Cells are ready for use on day 11.
  2. Aspirate media in each well from the culture plates, and add lysis buffer to each (50 μl for 96 well plate, and 200 μl for a 24 well plate).  Place the plate on the rocker for 15 min at room temperature.
  3. Dilute an aliquot of 4-Methylumbelliferyl substrate stock solution 40-fold in assay buffer, such that the final concentration of the substrate is 0.5 mg/ml.  This will be termed the “working solution”.
    1. Approximately 100 μl of working solution will be needed per sample.  Allow solution to equilibrate in 37 °C water bath.
    2. For each form of chitinase being tested there is a unique substrate. Separate assay plates will needed for each substrate.
  4. Prepare Methylumbelliferone standards by diluting the top Methylumbelliferone standard 1:100 (500 μg/ml), 1:1,000 (50 μg/ml), and 1:10,000 (5 μg/ml) in stop buffer.
    1. For best resolution, add 2 μl top Methylumbelliferone standard to 198 μl of stop buffer for the 1:100 dilution, followed by a 1:10 dilution series.
    2. Samples will be diluted further in the assay plate to yield 1,000 ng, 500 ng, 100 ng, and 10 ng (Figure 1).
  5. Load the standard wells in triplicate to the flat bottom black 96-well plate as demonstrated in Figure 1.


    Figure 1. Plate set-up for chitinase assay.  Red wells indicate 4-Methylumbelliferone standards, Purple indicates the sample blank (Working Solution), Green represents the positive control, and blue wells indicate sample wells.
  6. Dilute the chitinase positive control 1:200 in PBS to yield a final concentration of 1 μg/ml and load positive control wells with 10 μl chitinase and 90 μl working solution
  7. Use 100 μl working solution as the sample blank
  8. Add 90-99 μl working solution to each sample well in triplicate followed by 1-10 μl of each sample (samples are in lysis buffer)
    1. The amount of sample used for this assay will need to be optimized.  Some samples may contain so much chitinase activity that the fluorescence will be saturated.  This will vary considerably depending on the amount of cells plated and the chitinase activity of those cells.
  9. Wrap plate in foil and incubate at 37 °C for 30 min
    1. Incubation time may also need to be optimized. Cell lysates with high chitinase activity can be incubated for as little as 15 min. Alternatively, samples may be incubated for up to 1 h.
  10. Add 200 μl of stop buffer to each well to stop the reaction.
  11. Fluorescence can be measured on a plate reader at an excitation of 360 nm and emission of 450 nm within 30 min.
  12. Chitinase activity is calculated from the standard curve.  Alternatively, chitinase activity may be calculated  using the following equation:
    Units/mL= (Fluoresencesample – Fluoresenceblank) x 1.9 x 0.3 x Dilution Factor
    Fluoresence100 ng standard  x reaction time x sample Volume

Recipes

  1. Assay buffer
    Phosphate-Citrate Buffer pH=5.2  (26.7 ml of 0.2 M diabasic Sodium Phosphate, 23.3 ml of 0.1 M Citric Acid, top up to 100 ml DI water)
  2. Stop buffer
    Glycine-NaOH buffer pH = 10.6 (combine 25 ml 0.2 M glycine stock solution with 22.75 ml 0.2 M NaOH, and dilute with DI water to make a 100 ml solution)
  3. Protein lysis buffer  
    50 mM Tris HCl (pH 7.5)
    200 mM NaCl
    10% Glycerol
    0.5% TX-100
    1 mM DTT (added to buffer fresh, just before adding to cultures)
  4. Substrate Stock
    Prepare 20 mg/ml 4-Methylumbelliferyl substrate in DMSO.  
  5. Positive control
    Prepare 0.2 mg/ml chitinase from Trichoderma in PBS.

Acknowledgments

This protocol is adapted from Nance et al. (2012).

References

  1. Nance, J. P., Vannella, K. M., Worth, D., David, C., Carter, D., Noor, S., Hubeau, C., Fitz, L., Lane, T. E., Wynn, T. A. and Wilson, E. H. (2012). Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog 8(11): e1002990.   

材料和试剂

  1. 要分析的细胞。 例如来自C57BL/6小鼠的骨髓衍生的巨噬细胞
  2. L细胞
  3. cDMEM/F12
  4. 4-甲基伞形酮标记的底物:
    1. 4-甲基伞形酮N-乙酰基-β-D-氨基葡糖苷(外切蛋白酶活性;β-N-乙酰氨基葡糖苷酶活性)(Sigma-Aldrich,目录号:M2133)
    2. 4-甲基伞形酮N,N'-二乙酰基-β-D-壳寡糖苷(外切壳多糖酶活性;壳寡糖苷酶活性)(Sigma-Aldrich,目录号:M9763)
    3. 4-甲基伞形基β-D-N,N',N" - 三乙酰基壳三糖(内切粘附酶活性)(Sigma-Aldrich,目录号:M5639)
  5. 来自绿色木霉的几丁质酶(Sigma-Aldrich,目录号:C6242)
  6. 甲基伞形酮标准品(50mg/ml)(Sigma-Aldrich,目录号:M1381)
  7. PBS
  8. DMSO
  9. 读板器
  10. 碱式磷酸钠
  11. 柠檬酸
  12. 甘氨酸
  13. DTT
  14. 测试缓冲区(参见配方)
  15. 停止缓冲区(参见配方)
  16. 蛋白裂解缓冲液(参见配方)
  17. 基材库(见配方)
  18. 正控制(参见配方)

设备

  1. 平底黑色96孔板
  2. 荧光平板读数器(Molecular Devices)

程序

  1. 要分析的单元格。 我们使用来自C57BL/6小鼠的骨髓衍生的巨噬细胞。
    1. 简言之,用补充有20%L-细胞上清液(第0天)的10ml完全DMEM/F12从6-8周龄小鼠的胫骨和股骨冲洗骨髓。  
    2. 将骨髓细胞接种在非组织培养处理的10cm培养皿中,每个培养皿〜10ml细胞。 4天后,加入10ml另外的培养基。  
    3. 在第7天通过温和刮擦收获细胞。
    4. 将巨噬细胞以50,000细胞/孔接种在96孔板中的10%L-细胞上清液中的cDMEM/F12中,并允许静置3天。
    5. 在第10天,将培养基更换为不含L-细胞上清液的cDMEM/F12,并静置过夜。  
    6. 单元格可以在第11天使用。
  2. 从培养板的每孔中吸出培养基,并加入裂解缓冲液(对于96孔板为50μl,对于24孔板为200μl)。 将板在摇床上在室温下放置15分钟。
  3. 在测定缓冲液中将4-甲基伞形酮底物储备溶液的等分试样稀释40倍,使得底物的终浓度为0.5mg/ml。 这将被称为"工作解决方案"。
    1. 每个样品需要大约100μl的工作溶液。 使溶液在37℃水浴中平衡
    2. 对于每种形式的测试的几丁质酶,存在独特的底物。 每个底物需要单独的测定板
  4. 通过在终止缓冲液中稀释顶部Methylumbelliferone标准品1:100(500μg/ml),1:1,000(50μg/ml)和1:10,000(5μg/ml)制备Methylumbelliferone标准品。
    1. 为了获得最佳分辨率,将2μl上面的Methylumbelliferone标准品加入198μl1:100稀释的终止缓冲液中,然后加入1:10稀释系列。
    2. 样品将在测定板中进一步稀释以产生1,000ng,500ng,100ng和10ng(图1)。
  5. 将标准孔一式三份加载到平底黑色96孔板,如图1所示。


    图1.几丁质酶测定的板设置。红色孔表示4-甲基伞形酮标准品,紫色表示样品空白(工作溶液),绿色表示阳性对照,蓝色孔表示样品孔。
  6. 在PBS中稀释几丁质酶阳性对照1:200,得到终浓度为1μg/ml,并加入10μl几丁质酶和90μl工作溶液的阳性对照孔。
  7. 使用100μl工作溶液作为样品空白
  8. 向每个样品孔中加入90-99μl工作溶液,一式三份,然后加入1-10μl每种样品(样品在裂解缓冲液中)
    1. 用于此测定的样品量需要优化。一些样品可能含有如此多的几丁质酶活性,荧光会饱和。这将根据所接种的细胞的量和那些细胞的几丁质酶活性而显着变化。
  9. 将膜包裹在箔中并在37℃孵育30分钟
    1. 孵育时间也可能需要优化。 具有高几丁质酶活性的细胞裂解物可以孵育少至15分钟。 或者,可将样品孵育至多1小时。
  10. 向每个孔中加入200μl终止缓冲液以终止反应
  11. 荧光可以在板读数器上在360nm的激发和450nm的发射在30分钟内测量
  12. 几丁质酶活性由标准曲线计算。 或者,可以计算几丁质酶活性, 使用以下公式:
    单位/mL =(荧光样品 - 荧光空白)×1.9×0.3×稀释因子
    荧光 100 ng standard x反应时间x样品体积

食谱

  1. 测定缓冲区
    磷酸盐 - 柠檬酸盐缓冲液pH = 5.2  (26.7ml 0.2M磷酸二氢钠,23.3ml 0.1M柠檬酸,加至100ml去离子水)
  2. 停止缓冲区
    甘氨酸-NaOH缓冲液pH = 10.6(结合25ml 0.2M甘氨酸储备溶液与22.75ml 0.2M NaOH,并用去离子水稀释成100ml溶液)
  3. 蛋白裂解缓冲液
    50mM Tris HCl(pH7.5) 200 mM NaCl
    10%甘油
    0.5%TX-100
    1mM DTT(加入到缓冲液中,刚加入培养物之前)
  4. 基板库
    准备20毫克/毫升4-甲基伞形基底在DMSO中。  
  5. 正控制
    在PBS中制备来自木霉属的0.2mg/ml几丁质酶。

致谢

该协议改编自Nance等人(2012)。

参考文献

  1. Nance,JP,Vannella,KM,Worth,D.,David,C.,Carter,D.,Noor,S.,Hubeau,C.,Fitz,L.,Lane,TE,Wynn,TA和Wilson,EH 2012)。 几丁质酶依赖性控制大脑中的原生动物囊肿负荷 /em> 8(11):e1002990。   
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Worth, D., Nance, J. P. and Wilson, E. H. (2013). Chitinase Assay from Cultured Bone Marrow Derived Macrophages. Bio-protocol 3(23): e983. DOI: 10.21769/BioProtoc.983; Full Text
  2. Nance, J. P., Vannella, K. M., Worth, D., David, C., Carter, D., Noor, S., Hubeau, C., Fitz, L., Lane, T. E., Wynn, T. A. and Wilson, E. H. (2012). Chitinase dependent control of protozoan cyst burden in the brain. PLoS Pathog 8(11): e1002990.




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