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59Fe Uptake Assays in Paracoccidioides Species
副球孢子菌属的59Fe摄取实验   

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Abstract

Iron is an essential micronutrient required for virtually all organisms. This fact is related to the ability of the transition metal to exist in two oxidation states, the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+). Given the relative availability of aqueous iron (the element which constitutes ~5% of the earth’s crust) one is not surprised that iron is the most common prosthetic element in biology. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA) (Kosman, 2013). In this way, the uptake of iron in the absence or presence of the reducing agent ascorbic acid can be investigated by 59Fe uptake assays, as previously described (Eide et al., 1992). In the presence of ascorbic acid, the reductive-independent 59Fe uptake route is investigated. On the other hand, in the absence of ascorbic acid, the reductive-dependent 59Fe uptake route is stimulated. Using this strategy for the human pathogenic fungus Paracoccidioides species, the results showed that iron uptake by Pb01 in the absence of ascorbic acid was low, unlike what was observed for Pb18. These results suggest that only in Pb18 the iron uptake pathway is coupled to a ferric reductase (Bailão et al., 2015). In this protocol, we describe how to perform 59Fe uptake assays in Paracoccidioides species.

Keywords: Reductive iron assimilation pathway(还原铁同化途径), Reductive-independent iron uptake route(还原性独立铁吸收途径), Ascorbic acid(抗坏血酸), Quench buffer(淬火液), Gamma counter(γ计数)

Materials and Reagents

  1. Conical tube
  2. Wide-mouth screw-cap high-density polyethylene (scintillation) vials
  3. 13 x 100 mm test tubes
  4. Petri dish
  5. Filter type A/C 25 mm glass fiber filters from Pall Gelman
  6. 12 x 75 mm glass test tubes
  7. Wooden applicator (a thin wooden stick that is used in this protocol to put the filters containing radioactive cells inside the test tubes that will be read by the G counter)
  8. Disposable vacuum filtration system (TPP, catalog number: 99950 )
  9. Paracoccidioides spp. cells
  10. Culture media
    1. Liquid Brain Heart Infusion Broth (BHI) (Sigma-Aldrich, catalog number: 53286 ) with 4% glucose
    2. Liquid Synthetic Complete (SC medium) (see Recipes)
  11. Deionized water
  12. Yeast Nitrogen Base (YNB) without amino acids (BD, DifcoTM, catalog number: 291940 )
  13. Glucose (VWR, catalog number: 101175P )
  14. Amino acids (Sigma-Aldrich)
    ADE, TRP, HIS, ARG, MET, TYR, ISOLEU, LYS, PHE, GLU. ACID, ASP. ACID, VAL, THR, SER, URA, LEU
  15. Trypan blue solution, 0.4% for microscopy (Sigma-Aldrich, catalog number: 93595 )
  16. Ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 )
  17. Succinic acid (Sigma-Aldrich, catalog number: 398055 )
  18. Tris
  19. NaOH
  20. Analar glucose
  21. MES buffer (Sigma-Aldrich, catalog number: M3671 )
  22. Sodium citrate
  23. 59Fe (Perkin-Elmer, catalog number: NEZ037001MC )
  24. 1 M ascorbic acid (0.176 g in 1 ml sterilized water)
  25. Nitric acid (HNO3)
  26. Bathophenanthrolinedisulfonic acid (BPS) (Sigma-Aldrich, catalog number: B1375 )
  27. Iron (III) chloride (Sigma-Aldrich, catalog number: 157740 )
  28. SC complete media (see Recipes)
  29. Amino acids solution (see Recipes)
  30. Amino acid mix stock (see Recipes)
  31. 59Fe stock solutions (see Recipes)
  32. 10x quench buffer (see Recipes)
  33. Citrate uptake buffer (see Recipes)
  34. Acid-wash glassware treatment (see Recipes)

Equipment

  1. Shaker
  2. Hemocytometer
  3. Microfine forceps
  4. Glassware (erlenmeyers, bottle with blue cap, beakers, cylinders)
  5. Magnetic stirrer
  6. pH meter
  7. Vortex
  8. G counter (LKB Wallac Compu Gamma) with plastic g counting tube carriers
  9. Vacuum filtration manifold (Merck Millipore, model: 1225 )
  10. Source of vacuum, either standard lab pump or water aspirator
  11. Autoclave

Procedure

  1. Grow the Paracoccidioides spp. cells (from a stock* grown in BHI with 4% glucose medium for at least 72 h at 36 °C) in 200 ml BHI with 4% glucose medium for 72 h at 36 °C with shaking (around 300 rpm). After this, collect 5 x 106 viable cells counted in a hemocytometer with Trypan blue solution** and transfer to 30 ml of SC medium with no supplementation or supplemented with 200 μM BPS or 10 μM FeCl3 for 24 h at 36 °C with shaking.
    *Note: This stock remain viable until seven days. After this, a new culture has to be initiated in tubes containing fresh BHI with 4% glucose medium.
    ** Note: To count the viable cells, dilute the cells in 0.9% NaCl solution 100 times (it could be two 1:10 serial dilutions). After the dilution, get 10 μl of the cells and mix with 10 μl of Trypan blue solution. This corresponds to a 1:200 final dilution. Get 10 μl of this mixture and count the cells using a hemocytometer. The viable cells will not uptake the blue dye and will stay colorless.
  2. Collect 20 ml of cells into a conical tube by centrifugation at 1,200 x g for 10 min at 4 °C and wash cells once with 1 mM EDTA in citrate uptake buffer.
  3. Wash cells twice with citrate uptake buffer (pH 6.0).
  4. Re-suspend cells to a final volume of 15 ml with citrate uptake buffer.
  5. Transfer 7 ml of cells into a scintillation vial. You’ll need more than 7 ml of cells if you have more than two time points.
  6. Incubate cells for 15 min at 36 °C with shaking prior to addition of 59Fe.
  7. While shaking cells, take an aliquot (100 μl) from each scintillation vial and count the viable cells by a hemocytometer with Trypan blue solution.
    Note: This step is important to data normalization at the end of the experiment, since the data will be presented as pmol 59Fe/106 viable cells/time.
  8. For reductase-independent 59Fe uptake, add 140 μl 1 M ascorbic acid in distilled H2O to each scintillation vial (20 mM final concentration).
    Note: The ascorbic acid solution should be made just prior to use.
  9. To begin uptake, add 70 μl of 20 μM 59Fe solution to each of the scintillation vials.
  10. At each time point (be sure to have a zero time point), stop shaker, carefully remove the scintillation vials, and pipet 1 ml of cells into 13 x 100 mm test tubes containing 3 ml of ice-cold 1x quench buffer on ice. Do this in triplicate for each time point (Figure 1).


    Figure 1. Quenching uptake samples. At each time point 1 ml samples are withdrawn from the uptake vials and quenched in 3 ml ice-cold quench buffer. It is important to add the quench buffer immediately after collecting 1 ml sample.

  11. For filtration and washing of cells, pre-soak filters in 1x quench buffer in a Petri dish.
  12. Using the microfine forceps, carefully place 12 filters on the manifold (hold only the outer edges of the filters) (Figure 2). Place the top on the manifold and tighten it down with the blue plastic screw. Apply vacuum to the filter manifold using cold water to form a vacuum.


    Figure 2. Placing filters on the Millipore manifold. Prior to initiating the 59Fe-uptake by your fungal samples, the filters are wetted in quench buffer and then placed on each of the manifold’s frits with help of tweezers. The top on the manifold is then placed and tighten it down with the blue plastic screw. Apply vacuum to the filter manifold using cold water to form a vacuum.

  13. When all the samples have been collected, wash quenched samples on the Millipore manifold (Figure 3).


    Figure 3. Filtering quenched cell samples. After collecting and quenching all of the uptake samples, they are filtered and washed on the manifold for three times. It is important to wash filters in a series of # 1-12. Do not wash filter # 1 three times and then proceed to wash # 2 three times. If collecting more than the number of wells on the manifold, the first 12 filter set is removed for counting and replaced with fresh filters to continue the washing of additional samples.

  14. Pour samples over each filter. Rinse the test tube once with 3 ml ice-cold 1x quench buffer. Pour that over each corresponding filter. Rinse each filter three times with 3 ml ice-cold 1x quench buffer. Wash filters in a series of 1-12. For example, do not wash filter in position 1 three times and then proceed to wash the filter in position 2 three times. Leave manifold under vacuum until you have filtered and washed all samples.
  15. Leaving the manifold under vacuum, allow filters to dry (~30 sec) after the last wash.
  16. Using the forceps, remove each filter by folding it in on itself (NEVER touch the middle or top of the filter) and placing it in a 12 x 75 mm glass test tube.
  17. Continue with steps 11-15 until all of the samples have been processed.
  18. Using a wooden applicator, gently push the filters to the bottom of the test tube.
  19. Place the test tubes in the g counter carrier racks and count samples including 59Fe standards prepared in 12 x 75 mm test tubes. Use at least five points for standards, such as: 20 μM, 10 μM, 1 μM, 0.1 μM and 0.05 μM (Figure 4).
  20. Dispose of radioactive waste as per institutional environmental health and safety regulations.

Representative data



Figure 4. Representative data expected to obtain after the g counter reads. The standards are important to provide a calibration curve from which it will be possible to convert counts per minute (Cpm) to pmol 59Fe (top graphic). After normalization of the data with the 0 time point (fifth column), the pmol 59Fe is calculated based on the calibration curve and then it is possible to correlate the quantity of 59Fe (in pmol) 106 cells could internalize at a time, in this case 60 min (lower graphic).

Recipes

  1. SC complete media
    6.7 g YNB to 750 deionized water
    100 ml of 20% glucose
    100 ml of amino acid solution
    Sterile deionized water to 1,000 ml total volume
    Filter sterilize media
  2. Amino acid solution
    2.26 g of amino acid mix stock to 100 ml of deionized water
    Heat and stir into solution
    Allow to cool
  3. Amino acid mix stock
    2 g ADE
    10 g TRP
    10 g HIS
    7.5 g ARG
    2.5 g MET
    1.5 g TYR
    2.5 g ISOLEU
    2.5 g LYS
    2.5 g PHE
    5 g GLU. ACID
    5 g ASP. ACID
    7.5 g VAL
    10 g THR
    20 g SER
    2 g URA
    22.5 g LEU
    Note: This is a powder stock. The amino acid solution is explained above.
  4. 59Fe stock solutions
    1. To make a working stock of 20 μM 59Fe in ddH2O:
      For an experiment with 2 time points and 6 samples (individual vials), a minimum of 420 μl of 20 μM 59Fe is needed since each sample (vial) needs 70 μl of 20 μM 59Fe. It is recommended that the amount of 59Fe solution to be used is calculated assuming an extra 1.5 samples. For this example, prepare 7.5 x 70 μl, or 525 μl of 59Fe stock. This precaution ensures that, even with pipetting errors, there will be enough to make standards.
      Thus, for 525 μl of 20 μM 59Fe = 3.03 μl of 3.47 mM 59Fe stock (see Note below) plus 521.97 μl ddH2O. In each vial, therefore, 70 μl of 20 μM 59Fe stock in 7 ml uptake buffer = 0.2 μM 59Fe.
      Make serial dilutions of the 20 μM 59Fe stock to make the 2 and 0.2 μM stocks used for the standards.
      For example, 100 μl 20 μM 59Fe + 900 μl ddH2O = 2 μM 59Fe.
      Notes:
      1. Do not dilute the entire stock vial of radionuclide received from the vendor! Make up stock working solution only as needed.
      2. Calculating the [59Fe] in the radionuclide received from the vendor. Example is given for product number NEZ037001MC from Perkin Elmer Life Sciences.
    2. Divide the concentration by the specific activity. This gives the concentration in mg/ml [(mCi/ml)/(mCi/mg) = mg/ml].
      For example, If Perkin Elmer ships 0.5 mCi 59Fe having a specific activity of 72.87 mCi/mg with a concentration of 49.36 mCi/ml, the [59Fe] is determined in the following way:
      (49.36 mCi/ml)/(72.87 mCi/mg) = 0.67737 mg/ml
      (0.67737 mg/ml)/(59 g) = 0.01148 mmoles of Fe/ml
      (0.01148 mmol/ml) (1,000 ml/L) = 11.48 mM = [59Fe]
      This concentration is as of the stock date (the date the batch of 59Fe was made). In order to determine the concentration as of the date of your experiment, count the number of days between the stock date and your experiment date. Using an 59Fe decay table, find the corresponding decay factor. Multiply the decay factor by the stock date calibration. This gives you the [59Fe] in the stock vial as of the date of your experiment.
      To determine the volume of liquid in the 59Fe stock vial, divide the amount of iron you ordered (i.e., 500 μCi) by the concentration (mCi/ml) on the date one week after the iron was shipped.
  5. 10x quench buffer (1,000 ml)
    Note: Do not filter sterilize.
    44.4 g succinic acid (Final concentration = 375 mM)
    75.6 g Tris (Final concentration = 625 mM)
    37.5 g EDTA (acid form) (Final concentration = 128 mM)
    Everything will not go into solution until all three chemicals have been added and pH is adjusted.
    Adjust pH to 6.0 using NaOH
    Add ddH2O to final volume of 1,000 ml
    Store at 4 °C in acid-washed glassware
    Dilute to 1x for use in uptakes
  6. Citrate uptake buffer (1,000 ml)
    Note: Make and filter sterilize this buffer in acid-washed glassware.
    20.0 g Analar glucose (Final concentration = 2%)
    19.8 g MES buffer (Final concentration = 0.1 M)
    5.9 g Na-Citrate (Final concentration = 20 mM)
    Add NaOH to adjust pH to 6.0
    Add ddH2O to final volume of 1,000 ml
    Filter sterilize
    Store at room temperature
  7. Acid-wash glassware treatment
    Soak glassware in HNO3 (3-6 h)
    Note: This process could be performed overnight.
    Rinse 3 times in deionized water
    Fill 1/3 of the glassware with water and autoclave (cap with aluminum foil)
    Pour out water and wash more 3 times with water
    Refill glassware with water (1/3 full) and autoclave again (cap with aluminum foil)
    Pour water out just prior to use

Acknowledgments

The authors would like to thank to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Comissāo de Avaliação de Pessoal de Nível Superior (CAPES) and Fundação de Amparo a Pesquisa do Estado de Goiás (FAPEG). Development of this protocol was supported by a grant from the National Institutes of Health (US) DK053820 to DJK.

References

  1. Bailão, E. F. L. C., Lima, P. S., Silva-Bailão, M. G., Bailão, A. M., Fernandes. G. R., Kosman, D. J. and Soares, C. M. A. (2015). Paracoccidioides spp. ferrous and ferric iron assimilation pathways. Front Microbiol 6: 821.
  2. Eide, D., Davis-Kaplan, S., Jordan, I., Sipe, D. and Kaplan, J. (1992). Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently. J Biol Chem 267(29): 20774-20781.
  3. Kosman, D. J. (2013). Iron metabolism in aerobes: managing ferric iron hydrolysis and ferrous iron autoxidation. Coord Chem Rev 257(1): 210-217.

简介

铁是几乎所有生物体所必需的微量营养素。这一事实涉及过渡金属以两种氧化态存在的能力,即还原的亚铁(Fe 2+)和氧化的铁(Fe 3++)。考虑到铁水(构成地壳的〜5%的元素)的相对可用性,人们不惊讶的是,铁是生物学中最常见的假体元素。通常,真菌可以通过受体介导的铁载体或血红素的内化,和/或还原铁同化(RIA)吸收铁(Kosman,2013)。以这种方式,如上所述,可以通过59 Fe吸收测定来研究在还原剂抗坏血酸存在或不存在下铁的吸收(Eide等人)。 ,1992)。在抗坏血酸存在下,研究还原非依赖性59 Fe吸收途径。另一方面,在不存在抗坏血酸的情况下,刺激还原依赖性59 Fe吸收途径。使用这种策略用于人类致病真菌Paracoccidioides物种,结果表明,在没有抗坏血酸的情况下,通过 01的铁摄取是低的,不同于在 > Pb 18。这些结果表明,只有在Fe 18中,铁摄取路径与铁还原酶偶联(Bailão等人,2015)。在该方案中,我们描述了如何在Paracoccidioides物种中进行 59 Fe摄取测定。

关键字:还原铁同化途径, 还原性独立铁吸收途径, 抗坏血酸, 淬火液, γ计数

材料和试剂

  1. 圆锥管
  2. 宽口螺旋盖高密度聚乙烯(闪烁)小瓶
  3. 13×100mm试管
  4. 培养皿
  5. 来自Pall Gelman的过滤器型A/C 25mm玻璃纤维过滤器
  6. 12 x 75 mm玻璃试管
  7. 木制涂抹器(在本协议中用于将含有放射性细胞的过滤器放在试管内的薄木棒,将由G计数器读取)
  8. 一次性真空过滤系统(TPP,目录号:99950)
  9. Paracoccidioides 单元格
  10. 文化媒体
    1. 具有4%葡萄糖的液体脑心浸液肉汤(BHI)(Sigma-Aldrich,目录号:53286)
    2. 液体合成完全(SC介质)(参见配方)
  11. 去离子水
  12. 没有氨基酸的酵母氮基(YNB)(BD,Difco TM ,目录号:291940)
  13. 葡萄糖(VWR,目录号:101175P)
  14. 氨基酸(Sigma-Aldrich)
    ADE,TRP,HIS,ARG,MET,TYR,ISOLEU,LYS,PHE,GLU。 ACID,ASP。 ACID,VAL,THR,SER,URA,LEU
  15. 台盼蓝溶液,0.4%用于显微镜(Sigma-Aldrich,目录号:93595)
  16. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E9884)
  17. 琥珀酸(Sigma-Aldrich,目录号:398055)
  18. Tris
  19. NaOH
  20. 分析葡萄糖
  21. MES缓冲液(Sigma-Aldrich,目录号:M3671)
  22. 柠檬酸钠
  23. < sup> 59(Perkin-Elmer,目录号:NEZ037001MC)
  24. 1M抗坏血酸(0.176g,在1ml无菌水中)
  25. 硝酸(HNO 3)
  26. 碾红菲咯啉二磺酸(BPS)(Sigma-Aldrich,目录号:B1375)
  27. 氯化铁(III)(Sigma-Aldrich,目录号:157740)
  28. SC完整媒体(见配方)
  29. 氨基酸溶液(参见配方)
  30. 氨基酸混合物(见配方)
  31. 59 Fe储备溶液(见配方)
  32. 10x淬火缓冲液(参见配方)
  33. 柠檬酸摄取缓冲液(参见配方)
  34. 酸洗玻璃器皿处理(见配方)

设备

  1. 振动器
  2. 血细胞计数器
  3. 微细钳子
  4. 玻璃器皿(erlenmeyers,带蓝色瓶盖的瓶子,烧杯,圆筒)
  5. 磁力搅拌器
  6. pH计
  7. 涡流
  8. G计数器(LKB Wallac Compu Gamma),带塑料计数管支架
  9. 真空过滤歧管(Merck Millipore,型号:1225)
  10. 真空源,标准实验室泵或吸水器
  11. 高压灭菌

程序

  1. 种植 Paracoccidioides spp。细胞(来自于在4%葡萄糖培养基中在4%葡萄糖培养基中在4%葡萄糖培养基中生长至少72小时的库存)在具有4%葡萄糖培养基的200ml BHI中在36℃振荡(约300rpm)72小时。之后,收集在具有台盼蓝溶液**的血细胞计数器中计数的5×10 6个活细胞**,并转移至30ml未补充或补充有200μMBPS或10μMFeCl 2的SC培养基> 3℃,在36℃下摇动24小时 *注意:这个股票一直存活直到七天。之后,必须在含有含4%葡萄糖培养基的新鲜BHI的管中开始新的培养。
    注意:为了计数活细胞,将细胞在0.9%NaCl溶液中稀释100次(可以是两次1:10连续稀释)。稀释后,得到10微升的细胞,并与10微升的台盼蓝溶液混合。这对应于1:200的最终稀释。获得10微升的混合物,并使用血细胞计数细胞。活细胞不会吸收蓝色染料,并保持无色。
  2. 通过在4℃下以1,200×g离心10分钟将20ml细胞收集到锥形管中,并用在柠檬酸盐吸收缓冲液中的1mM EDTA洗涤细胞一次。
  3. 用柠檬酸盐摄取缓冲液(pH 6.0)洗涤细胞两次
  4. 用柠檬酸盐吸收缓冲液重悬细胞至最终体积为15ml
  5. 转移7毫升的细胞到闪烁瓶。如果你有两个以上的时间点,你需要超过7毫升的细胞
  6. 孵育细胞15分钟,在36℃下摇动,然后加入59 Fe。
  7. 在摇动细胞的同时,从每个闪烁瓶中取出等分试样(100μl),并通过血细胞计数器用台盼蓝溶液计数活细胞。
    注意:这个步骤对于实验结束时的数据标准化很重要,因为数据将以pmol/sup/6活细胞/时间表示。 。
  8. 对于还原酶非依赖性的59 Fe吸收,将在蒸馏H 2 O中的140μl1M抗坏血酸加入到每个闪烁管中(20mM终浓度)。
    注意:抗坏血酸溶液应在使用前立即进行。
  9. 开始吸收,向每个闪烁瓶中加入70μl20μM的 Fe溶液。
  10. 在每个时间点(确保具有零时间点),停止振荡器,小心地移除闪烁瓶,并将1ml细胞吸入含有3ml冰冷的1×淬灭缓冲液的13×100mm试管中。对每个时间点重复三次(图1)。


    图1.淬灭摄取样品。在每个时间点,从吸收管中取出1ml样品,并在3ml冰冷的淬灭缓冲液中淬灭。在收集1ml样品后立即添加淬灭缓冲液很重要。

  11. 对于细胞的过滤和洗涤,在培养皿中的1x淬灭缓冲液中预先浸泡过滤器
  12. 使用微型镊子,小心地在歧管上放置12个过滤器(只保持过滤器的外边缘)(图2)。将顶部放在歧管上,用蓝色塑料螺丝拧紧。使用冷水对过滤器歧管施加真空以形成真空。


    图2.在Millipore歧管上放置过滤器。在启动真菌样品的Fe吸收之前,将过滤器在淬灭缓冲液中润湿,然后置于每个歧管的玻璃料 帮助镊子。然后放置歧管上的顶部,并用蓝色塑料螺钉将其拧紧。使用冷水对过滤歧管施加真空以形成真空
  13. 收集所有样品后,在Millipore歧管上冲洗淬火的样品(图3)

    图3.过滤淬灭的细胞样品。在收集和淬灭所有吸收样品后,将它们过滤并在歧管上洗涤三次。重要的是在#1-12系列中清洗过滤器。不要洗涤过滤器#1三次,然后继续洗涤#2三次。如果收集多于歧管上的孔数,则去除前12个过滤器组以进行计数,并用新的过滤器替换,以继续洗涤额外的样品。
  14. 将样品倒在每个过滤器上。用3ml冰冷的1×淬灭缓冲液冲洗试管一次。将其倒在每个相应的过滤器上。用3ml冰冷的1×淬灭缓冲液冲洗每个过滤器三次。洗涤过滤器系列1-12。例如,不要在位置1清洗过滤器三次,然后继续在位置2清洗过滤器三次。在真空下离开歧管,直到您过滤并洗涤所有样品
  15. 将歧管留在真空下,让过滤器在最后一次清洗后干燥(〜30秒)。
  16. 使用镊子,通过将每个过滤器折叠在自身上(请勿触摸过滤器的中间或顶部),并将其放置在一个12 x 75毫米的玻璃试管中。
  17. 继续执行步骤11-15,直到所有样品都已处理完毕。
  18. 使用木制涂抹器,轻轻地将过滤器推到试管底部
  19. 将试管放在g计数器托架中,并计数样品,包括在12 x 75 mm试管中制备的 59 Fe标准品。 对于标准品,至少使用5个点,例如:20μM,10μM,1μM,0.1μM和0.05μM(图4)。
  20. 按照制度环境健康和安全规定处置放射性废物

代表数据



图4. g计数器读数后期望获得的代表性数据标准品对于提供校准曲线很重要,从中可以将每分钟计数(Cpm)转换为pmol 59 Fe(top graphic)。 在用0时间点(第五列)归一化数据后,基于校准曲线计算pmol Fe,然后可以将 59 > Fe(以pmol)10 6个细胞可以一次内在化,在这种情况下为60分钟(下图)。

食谱

  1. SC完成媒体
    6.7g YNB至750去离子水
    100ml 20%葡萄糖 100ml氨基酸溶液
    无菌去离子水至1,000ml总体积
    过滤灭菌介质
  2. 氨基酸溶液
    2.26g氨基酸混合物料加到100ml去离子水中 加热并搅拌溶液
    允许冷却
  3. 氨基酸混合物
    2克ADE
    10 g TRP
    10 g HIS
    7.5克ARG
    2.5 g MET
    1.5克TYR
    2.5 g ISOLEU
    2.5克LYS
    2.5 g PHE
    5g GLU。 ACID
    5g ASP。 ACID
    7.5 g VAL
    10 g THR
    20克SER
    2 g URA
    22.5克LEU
    注意:这是一种粉末。氨基酸溶液如上所述。
  4. 59 Fe储备溶液
    1. 为了在ddH 2 O中制备20μM的工作原料: Fe Fe:
      对于具有2个时间点和6个样品(单个小瓶)的实验,需要最少420μl的20μM Fe,因为每个样品(小瓶)需要70μl的20μM 59 Fe。建议在假设额外的1.5个样品的情况下计算所使用的 Fe溶液的量。对于这个例子,准备7.5 x 70微升,或525微升 59 Fe股票。这种预防措施确保即使有移液错误,也足以制定标准 因此,对于525μl的20μM的59μLFe =3.03μl的3.47mM的59μL的Fe储备液(参见下面的注释)加上521.97μlddH 2 SO 4, O.因此,在每个小瓶中,70μl的在7ml摄取缓冲液中的20μM的59μLFe储备液=0.2μM 59 Fe。
      制备20μM 59 Fe原液的系列稀释液,使2和0.2μM原液用于标准品。
      例如,100μl20μM 59 Fe +900μlddH 2 O =2μM 59 Fe。 注意:
      1. 不要稀释从供应商收到的放射性核素的整个储存小瓶!只有在需要时才组成库存工作解决方案。
      2. 计算从供应商接收的放射性核素中的[ 59 Fe]。给出了来自Perkin Elmer Life Sciences的产品号NEZ037001MC的实例。
    2. 将浓度除以比活度。这得到以mg/ml [(mCi/ml)/(mCi/mg)= mg/ml]表示的浓度。
      例如,如果Perkin Elmer装有浓度为49.36mCi/ml的比活性为72.87mCi/mg的0.5mCi 59 Fe,则测定[ 59 Fe]按以下方式:
      (49.36mCi/ml)/(72.87mCi/mg)= 0.67737mg/ml
      (0.67737mg/ml)/(59g)= 0.01148mmol Fe/ml (0.01148mmol/ml)(1,000ml/L)= 11.48mM = [59] Fe]/该浓度是储存日期(制备 59的批次的日期)。为了确定实验日期的浓度,请计算库存日期和实验日期之间的天数。使用 59 Fe衰减表,找到相应的衰减因子。通过股票日期校准乘以衰减系数。这给你在实验日期之前的储存小瓶中的[ 59 Fe]。
      为了测定 59 Fe储液瓶中的液体体积,将所订购的铁量(即<500>,500μCi)除以浓度(mCi/ml)铁发货后一周的日期。
  5. 10x淬灭缓冲液(1000ml) 注意:不要过滤灭菌。
    44.4g琥珀酸(最终浓度= 375mM) 75.6g Tris(终浓度= 625mM) 37.5g EDTA(酸形式)(最终浓度= 128mM) 在添加所有三种化学品并调节pH之前,一切都不会进入溶液。
    用NaOH
    调节pH至6.0 将ddH <2> O加入到最终体积为1000ml
    在4°C下储存在酸洗玻璃器皿
    中 稀释为1x,用于摄入量
  6. 柠檬酸盐摄取缓冲液(1,000ml) 注意:在酸洗的玻璃器皿中制作并过滤灭菌此缓冲液。
    20.0g分析葡萄糖(终浓度= 2%)
    19.8g MES缓冲液(终浓度= 0.1M) 5.9g柠檬酸钠(终浓度= 20mM) 加入NaOH调节pH至6.0
    将ddH <2> O加入到最终体积为1000ml
    过滤灭菌
    在室温下贮存
  7. 酸洗玻璃器皿处理
    将玻璃器皿浸泡在HNO <3> (3-6小时)
    中 注意:此过程可以在隔夜进行。
    在去离子水中冲洗3次 用水和高压灭菌器(铝箔盖)填充玻璃器皿的1/3。
    倒出水,用水洗涤3次,
    将玻璃器皿加满水(1/3满),再次高压灭菌(用铝箔盖上) 在使用前倒出水

致谢

作者要感谢国家科学技术委员会(CNPq),法国国家高级委员会(CAPES)和基础设施委员会(FAPEG)。该方案的开发由来自DJK的美国国立卫生研究院(US)DK053820的资助支持。

参考文献

  1. Bailão,E.F.L.C.,Lima,P.S.,Silva-Bailão,M.G.,Bailão,A.M.,Fernandes。 GR,Kosman,DJ and Soares,CMA(2015)。  Paracoccidioides spp。亚铁和三价铁同化途径。 前微生物 6:821.
  2. Eide,D.,Davis-Kaplan,S.,Jordan,I.,Sipe,D。和Kaplan,J.(1992)。  酿酒酵母中铁摄取的调节 。铁氧还蛋白和Fe(II)转运蛋白是独立调节的。 J Biol Chem 267(29):20774-20781。
  3. Kosman,DJ(2013)。  aerobes中的铁代谢: 管理三价铁水解和亚铁自氧化。 Coord Chem Rev 257(1):210-217。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kosman, D. J., Bailão, E. L., Silva-Bailão, M. G. and Soares, C. d. (2016). 59Fe Uptake Assays in Paracoccidioides Species. Bio-protocol 6(18): e1930. DOI: 10.21769/BioProtoc.1930.
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