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Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues
小鼠组织转铁蛋白和非转铁蛋白结合铁摄取的测量   

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本实验方案简略版
Cell Metabolism
Jul 2015

Abstract

Iron in blood plasma is bound to its transport protein transferrin, which delivers iron to most tissues. In iron overload and certain pathological conditions, the carrying capacity of transferrin can become exceeded, giving rise to non-transferrin-bound iron, which is taken up preferentially by the liver, kidney, pancreas, and heart. The measurement of tissue transferrin- and non-transferrin-bound iron (TBI and NTBI, respectively) uptake in vivo can be achieved via intravenous administration of 59Fe-labeled TBI or NTBI followed by gamma counting of various organs. Here we describe a detailed protocol for the measurement of TBI and NTBI uptake by mouse tissues.

Keywords: NTBI (NTBI), TBI (TBI), 59Fe (59fe), Tissue iron uptake (组织铁的吸收), Iron overload (铁过载)

Materials and Reagents

  1. 1 ml TB syringe with 27-gauge needle (BD Biosciences, catalog number: 309623 )
  2. Plastic wrap
  3. 3.5” x 3.5” weigh boat (Thermo Fisher Scientific, Fisher Scientific, catalog number: 8732113 )
  4. 1.5-ml microcentrifuge tubes
  5. 0.45 µm filter unit (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-740-65B )
  6. AmiconTM Ultra-15 centrifugal filter units (Merck Millipore, catalog number: UFC903024 )
  7. 5 ml tube
  8. Adult mice (> 6 weeks, any strain) (e.g., Balb/cJ)
  9. 59FeCl3 (Perkin Elmer, catalog number: NEZ037500UC )
    Note: The minimum specific activity of 59Fe we have used to obtain decent signal from our gamma counter was 0.2 Ci/mmol at the concentration of ~3 µCi/µl, which gave whole body counts per minute of ~100,000 per each animal. Our gamma counting efficiency is about 10% for 59Fe.
  10. Nitrilotriacetic acid (NTA) (Sigma-Aldrich, catalog number: N9877-100G )
  11. HEPES (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC172570250 )
  12. Tris (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP152-5 )
  13. Sodium bicarbonate (NaHCO3) (Fisher Scientific, catalog number: S233-500 )
  14. Sodium citrate dihydrate (Thermo Fisher Scientific, Fisher Scientific, catalog number: S279-500 )
  15. Ferric chloride hexahydrate (FeCl3·6H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I88-100 )
  16. Hydrochloric acid (HCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A144-212 )
  17. Ethanol
  18. Human apo-transferrin (Sigma-Aldrich, catalog number: T2252 )
  19. PBS (VWR, Corning®, catalog number: 21-040-CM )
  20. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S2713 )
  21. FisherbrandTM absorbent underpads (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-206-64 )
  22. Radioactive decontaminating solution (Thermo Fisher Scientific, Fisher Scientific, catalog number: NC9633347 )
  23. 20 mM Fe-NTA (1:4) (see Recipes)
  24. Ferric citrate solution (see Recipes)
  25. 59Fe-labeled ferric citrate solution (see Recipes)
  26. 59Fe-labeled transferrin (see Recipes)
  27. 0.9% NaCl solution (see Recipes)

Equipment

  1. Gamma counter (PerkinElmer, catalog number: 2480-0010 )
    The program for 59Fe detection was set using the following parameters:
    1. Counting window: Dynamic-keV
    2. Peak position: 1292 keV
    3. Low boundary: 1020 keV
    4. High boundary: 1400 keV
    5. Threshold: 20%
    6. Max. assay deviation: 20%
    7. Max. normalization deviation: 50%
    8. Warning assay deviation: 15%
    9. Significant CPM/keV: 10
    10. Counting spectrum type: many peaks
  2. Gamma counting tubes (5 x 28 mm) and RIA racks (PerkinElmer, catalog number: 1480-151 )
    1. Prior to gamma counting, empty gamma counting tubes are measured to ensure no 59Fe contamination (i.e., < 25 CPM = background).
  3. Mouse restrainer (Braintree Scientific, catalog number: MTI STD )
  4. Basic surgical kit for small animals

Procedure

  1. For tissue NTBI uptake measurement, inject 0.1 ml of ferric citrate solution (containing 70 µg iron) to a mouse via tail vein to transiently saturate the iron-binding capacity of plasma transferrin.
    This step is required so that any subsequently injected iron will not bind to transferrin and hence will be non-transferrin-bound iron (NTBI). Return the mouse to the cage and wait for 10 min before going to step 2. See Figure 1 for a typical injection station setup.
    For tissue TBI uptake measurement, inject 0.1 ml of 59Fe-labeled transferrin (containing 150 µg transferrin) to a mouse via tail vein. Return the mouse to the cage and wait for 2 h before going to step 3.


    Figure1. Typical setup for tail vein injection station (A) and tissue harvesting station (B)

  2. Inject 0.1 ml of 59Fe-labeled ferric citrate solution (2 µCi) to the mouse via tail vein. Return the mouse to the cage.
  3. Two hours after injection of 59Fe-labeled ferric citrate solution (for NTBI uptake) or 59Fe-labeled transferrin (for TBI uptake), euthanize the mouse with an overdose of isoflurane or other proper methods and place the mouse on an absorbent pad. Cut the mouse tail off to remove residual 59Fe radioactivity that is retained in the tail (i.e., radioactivity that did not enter the circulation). See Figure 1 for a typical harvesting station setup.
  4. Sterilize the mouse abdomen and chest area with 70% ethanol. Open the abdominal cavity with blunt-end sterile scissors and collect tissues of interest (e.g., pancreas, spleen, liver, kidney, etc.). Then open the chest cavity to collect tissues such as lung and heart.
  5. Rinse tissue in 0.9% NaCl solution poured in a disposable weigh boat and remove excess liquid by dabbing the tissues on a stack of paper towels.
  6. Use plastic wrap to wrap each tissue briefly prior to transfer into a gamma counting tube to minimize cross contamination of 59Fe radioactivity between samples. Optionally, transfer tissue to a clean 1.5-ml microcentrifuge tube if the tissue sample can fit in it. Then measure the tissue radioactivity using a gamma counter by putting the 1.5-ml tube containing the tissue sample into a gamma counting tube rack (see Figure 2).
  7. For the mouse carcass, use plastic wrap to securely wrap the whole carcass prior to transfer into a gamma counting tube (see Figure 2).


    Figure 2. Illustration of radioactive tissue handling and loading to gamma counting tubes prior to measurement by a gamma counter. A. Materials required for tissue sample wrapping. B. Top view of the gamma counting rack with previously wrapped samples loaded in each gamma counting tube. C. The gamma counting rack containing the sample tube is placed on the rack belt of the gamma counter, ready to be loaded inside the gamma detector (left panel). The gamma counter screen showing real-time radioactivity being detected from the tissue sample (right panel).

  8. Measure organ and carcass 59Fe radioactivity by using a gamma counter. Calculate tissue NTBI and TBI uptakes as a percentage of whole-body counts per minute (CPM) (whole-body CPM = CPM values from all tissues + CPM value from carcass) (see Figure 3).


    Figure 3. Example of raw data and analysis of results. Two hours after injection with 59Fe-labeled ferric citrate, WT and Zip14 KO mice at 3 weeks of age were sacrificed and their tissues and carcasses were subjected to gamma counting. A. Counts per minute (CPM) values from each sample were recorded. Whole-body CPM values were calculated from the sum of all tissues and carcass CPMs. B. Tissue 59Fe accumulation is expressed as % of whole body and calculated as (tissue CPM/whole body CPM) x 100. C. Resultant figure from panel B is shown. Data were analyzed by using unpaired student’s t-test. Values are means ± SE (n = 3/group). ***P < 0.001, **P < 0.01.

Notes

After each experiment, use radioactive cleaning reagent to clean the surgical tools, mouse cages, and related equipment to decontaminate 59Fe. Perform a radioactive swipe test on the related equipment and area where the experiment is performed after each experiment to ensure no contamination is observed. Radioactive waste products should be disposed of by following your local Environmental Health and Safety (EH&S) regulations.

Recipes

  1. 20 mM Fe-NTA (1:4 molar ratio)
    20 mM HEPES/20 mM Tris buffer
    0.4 M NTA
    0.4 M FeCl3
    1. Prepare 20 mM HEPES/20 mM Tris buffer
      To 450 ml ddH2O, dissolve 2.38 g HEPES and 1.21 g Tris.
      Adjust pH to 6.0 with 10 N HCl.
      Bring final volume to 500 ml with ddH2O.
    2. Prepare 0.4 M NTA
      In 20 ml 20 mM HEPES/20 mM Tris buffer, add 4.28 g NTA.
      Adjust pH to 6.0 with 10 N NaOH by adding ~3 ml of 10 N NaOH at the beginning then drop-wise until pH reaches 6.0 (Caution: pH changes rapidly between pH 5-7).
      Bring final volume to 56 ml with 20 mM HEPES/20 mM Tris buffer.
    3. Prepare 0.4 M FeCl3 in 100 mM HCl
      To make 100 mM HCl, add 500 µl of 37% (equivalent to 12 M) HCl solution to 11.5 ml ddH2O.
      Dissolve 1.08 g FeCl3·6H2O in 10 ml 100 mM HCl.
    4. Prepare 20 mM Fe-NTA (1:4 molar ratio)
      In a 50 ml beaker with stir bar inside, add 10 ml of 20 mM HEPES/20 mM Tris buffer.
      Leave the pH electrode inside the solution and track the solution pH along the rest of the procedure.
      Add 2 ml of 0.4 M FeCl3 prepared in step 1c. The solution pH should now be ~2.
      Add 8 ml of 0.4 M NTA prepared in step 1b.
      Adjust to final pH of 6.95 with 100 mM NaOH.
      Bring final volume to 40 ml with 20 mM HEPES/20 mM Tris buffer.
      Filter sterilized the 20 mM Fe-NTA solution through 0.45 µm filter unit.
  2. Ferric citrate solution
    0.1 M citrate buffer
    12.5 mM FeCl3·6H2O
    1. Prepare 0.1 M citrate buffer
      To 40 ml ddH2O, dissolve 2.25 g Na3C6H5O7·2H2O (tri-sodium citrate dihydrate).
      Adjust pH to 6.6 with 10 M HCl.
      Bring final volume to 50 ml with ddH2O.
    2. For 50 ml stock, add 0.169 g FeCl3·6H2O to 50 ml 0.1 M citrate buffer.
  3. 59Fe-labeled ferric citrate solution
    0.1 M citrate buffer, 59FeCl3
    To 100 µl 0.1 M citrate buffer, add 2 µCi 59FeCl3.
    Mix well.
  4. 59Fe-labeled transferrin
    10 mM NaHCO3
    18.75 µM apo-transferrin
    20 mM Fe-NTA (1:4 molar ratio)
    59FeCl3
    1. To 50 ml PBS, add 0.042 g NaHCO3.
    2. Add 7.5 mg apo-transferrin to 5 ml NaHCO3 in PBS buffer prepared in step 4a.
    3. Add 2.5 µl 59FeCl3 to 12.5 µl of 20 mM Fe-NTA solution.
    4. Mix solutions prepared in steps 4b and 4c. Incubate at RT for 1 h to allow 59Fe to bind to transferrin.
    5. Remove unbound 59Fe by using AmiconTM Ultra-15 centrifugal filter unit.
      1. Add 4 ml of 59Fe-labeled transferrin prepared in step 4d to an AmiconTM Ultra-15 centrifugal filter unit.
      2. Centrifuge at 5,000 x g for 20 min at RT, discard eluate in a radioactive waste container.
      3. Add 4 ml 10 mM NaHCO3 (prepared in step 4a) to the filter unit and centrifuge at 5,000 x g for 20 min at RT, discard eluate.
      4. Repeat step eiii for 3 additional times. In last wash, you may wish to measure 59Fe radioactivity found in the eluate to ensure that the 59Fe not bound to TBI has been thoroughly removed. The eluate 59Fe radioactivity of greater than 30 CPM may indicate the requirement for additional washes.
      5. Add 4 ml of 10 mM NaHCO3 buffer on top of the filter unit and collect all the retentate (which contains the newly added 4 ml of 10 mM NaHCO3 and the 59Fe-labeled transferrin, making the collecting volume to ~4 ml) into a fresh 5 ml tube. Measure 59Fe CPM of the 59Fe-labeled transferrin solution to ensure sufficient radioactivity to be able to detect tissue-associated 59Fe from each animal (desirably ~10,000 CPM/100 µl 59Fe-labeled transferrin solution). Store at 4 °C until use for tail vein injection.
  5. 0.9% NaCl solution, autoclaved
    To every 1 L of ddH2O, add 9 g of NaCl.
    Autoclave to sterilize and store at 4 °C until use.

Acknowledgments

This work was supported by the National Institutes of Health grant DK080706 (to M.D.K.). The protocol was adapted from Craven et al. (1987), and a short version of the adapted protocol was published in Jenkitkasemwong et al. (2015).

References

  1. Craven, C. M., Alexander, J., Eldridge, M., Kushner, J. P., Bernstein, S. and Kaplan, J. (1987). Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc Natl Acad Sci U S A 84(10): 3457-3461.
  2. Jenkitkasemwong, S., Wang, C. Y., Coffey, R., Zhang, W., Chan, A., Biel, T., Kim, J. S., Hojyo, S., Fukada, T. and Knutson, M. D. (2015). SLC39A14 is required for the development of hepatocellular iron overload in murine models of hereditary hemochromatosis. Cell Metab 22(1): 138-150. 

简介

血浆中的铁结合其转运蛋白转铁蛋白,其将铁递送至大多数组织。 在铁过载和某些病理状况下,转铁蛋白的携带能力可能超过,导致非转铁蛋白结合的铁,其优先被肝脏,肾脏,胰腺和心脏摄取。 分别测量组织转铁蛋白和非转铁蛋白结合的铁(分别为TBI和NTBI)在体内的摄取可以通过静脉内施用59 Fe标记的TBI或 NTBI,然后是各种器官的γ计数。 在这里我们描述了测量小鼠组织的TBI和NTBI摄取的详细协议。

关键字:NTBI, TBI, 59fe, 组织铁的吸收, 铁过载

材料和试剂

  1. 带有27号针的1ml TB注射器(BD Biosciences,目录号:309623)
  2. 塑料包装
  3. 3.5"×3.5"称重舟(Thermo Fisher Scientific,Fisher Scientific,目录号:8732113)
  4. 1.5 ml微量离心管
  5. 0.45μm过滤器单元(Thermo Fisher Scientific,Fisher Scientific,目录号:09-740-65B)
  6. Amicon Super-15离心过滤器单元(Merck Millipore,目录号:UFC903024)
  7. 5 ml管
  8. 成年小鼠(> 6周,任何菌株)(例如,Balb/cJ)

  9. (Perkin Elmer,目录号:NEZ037500UC) 注意:在〜3μCi/μl的浓度下,我们用于从我们的γ计数器获得体面信号的 59 Fe的最小比活性为0.2Ci/mmol,这提供了全身计数 每分钟〜100,000每只动物。 对于 59 Fe,我们的伽马计数效率约为10%。
  10. 次氮基三乙酸(NTA)(Sigma-Aldrich,目录号:N9877-100G)
  11. HEPES(Thermo Fisher Scientific,Fisher Scientific,目录号:AC172570250)
  12. Amicon Super-15离心过滤器单元(Merck Millipore,目录号:UFC903024)
  13. 5 ml管
  14. 成年小鼠(> 6周,任何菌株)(例如,Balb/cJ)

  15. (Perkin Elmer,目录号:NEZ037500UC) 注意:在〜3μCi/μl的浓度下,我们用于从我们的γ计数器获得体面信号的 59 Fe的最小比活性为0.2Ci/mmol,这提供了全身计数 每分钟〜100,000每只动物。 对于 59 Fe,我们的伽马计数效率约为10%。
  16. 次氮基三乙酸(NTA)(Sigma-Aldrich,目录号:N9877-100G)
  17. HEPES(Thermo Fisher Scientific,Fisher Scientific,目录号:AC172570250)
    ...
  18. Human apo-transferrin (Sigma-Aldrich, catalog number: T2252)
  19. PBS (VWR, Corning®, catalog number: 21-040-CM)
  20. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S2713)
  21. FisherbrandTM absorbent underpads (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-206-64)
  22. Radioactive decontaminating solution (Thermo Fisher Scientific, Fisher Scientific, catalog number: NC9633347)
  23. 20 mM Fe-NTA (1:4) (see Recipes)
  24. Ferric citrate solution (see Recipes)
  25. 59Fe-labeled ferric citrate solution (see Recipes)
  26. 59Fe-labeled transferrin (see Recipes)
  27. 0.9% NaCl solution (see Recipes)

Equipment

  1. Gamma counter (Perkin Elmer, catalog number: 2480-0010)
    The program for 59Fe detection was set using the following parameters:
    1. Counting window: Dynamic-keV
    2. Peak position: 1292 keV
    3. Low boundary: 1020 keV
    4. High boundary: 1400 keV
    5. Threshold: 20%
    6. Max. assay deviation: 20%
    7. Max. normalization deviation: 50%
    8. Warning assay deviation: 15%
    9. 显着CPM/keV:10
    10. 计数谱类型:许多峰
  2. γ计数管(5×28mm)和RIA架(Perkin Elmer,目录号:1480-151)
    1. 在γ计数之前,测量空的γ计数管以确保没有 Fe污染(即,<25 CPM =背景)。
  3. 鼠标限制器(Braintree Scientific,目录号:MTI STD)
  4. 小动物基本手术包

程序

  1. 对于组织NTBI摄取测量,通过尾静脉向小鼠注射0.1ml柠檬酸铁溶液(含有70μg铁),以瞬时饱和血浆转铁蛋白的铁结合能力。
    该步骤是必需的,使得任何随后注射的铁不会结合转铁蛋白,因此将是非转铁蛋白结合铁(NTBI)。将鼠标放回笼中,等待10分钟,然后转到步骤2.请参见图1,了解典型的注射台设置。
    对于组织TBI摄取测量,通过尾静脉将0.1ml的59 Fe标记的转铁蛋白(含有150μg转铁蛋白)注射到小鼠。将鼠标放回笼子,等待2小时,然后转到步骤3

    图1。尾静脉注射部位(A)和组织收获部位(B)的典型设置

  2. 通过尾静脉向小鼠注射0.1ml的59 Fe标记的柠檬酸铁溶液(2μCi)。将鼠标放回笼子 />
  3. 在注射59 Fe标记的柠檬酸铁溶液(用于NTBI摄取)或59 Fe标记的转铁蛋白(用于TBI摄取)后两小时,用过量的安慰剂异氟烷或其它合适的方法,并将小鼠放在吸收垫上。切开小鼠尾部以除去保留在尾部的残留的 Fe放射性(即未进入循环的放射性)。参见图1,了解典型的收割站设置
  4. 用70%乙醇灭菌小鼠腹部和胸部。用钝端无菌剪刀打开腹腔,并收集感兴趣的组织(例如,胰腺,脾,肝,肾,等)。然后打开胸腔收集肺和心脏等组织。
  5. 在0.9%NaCl溶液中冲洗组织,将其倒入一次性称重舟中,并通过将一叠纸巾上的组织擦拭来除去多余的液体。
  6. 在转移到γ计数管之前,使用塑料包裹短暂包裹每个组织,以最小化样品之间的 Fe放射性的交叉污染。任选地,如果组织样品可以装入其中,则将组织转移至干净的1.5ml微量离心管。然后使用γ计数器通过将含有组织样品的1.5ml管置于γ计数管架中来测量组织放射性(参见图2)。
  7. 对于小鼠胴体,在转移到γ计数管之前,使用塑料包裹物将整个屠体牢固包裹(见图2)。


    图2.在通过γ计数器测量之前放射性组织处理和装载到γ计数管的图示。 A.组织样品包装所需的材料。 B.具有先前包裹的样品的γ计数架的顶视图装载在每个γ计数管中。 C.将包含样品管的γ计数架放置在γ计数器的齿条带上,准备装载在γ检测器(左图)内。伽玛计数器屏幕显示从组织样品检测的实时放射性(右图)
  8. 使用γ计数器测量器官和胴体 Fe放射性。计算组织NTBI和TBI以每分钟全身计数(CPM)的百分比(全身CPM =来自所有组织的CPM值+来自屠体的CPM值)(见图3)的百分比。

    图3.原始数据的实例和结果分析。注射后3小时,用Fe标记的柠檬酸铁,WT和Zip14 KO小鼠注射2小时后,处死并对它们的组织和屠体进行γ计数。记录来自每个样品的每分钟计数(CPM)值。从所有组织和尸体CPM的总和计算全身CPM值。 B.组织59 Fe累积表示为全身的%,并计算为(组织CPM /全身CPM)×100。显示来自图B的结果图。通过使用未配对的学生的测试来分析数据。值为平均值±SE(n = 3 /组)。 *** P 0.001,**


    P 0.01

笔记

每次实验后,使用放射性清洁剂清洁外科工具,小鼠笼和相关设备以净化 59 Fe。 在每次实验后进行实验的相关设备和区域进行放射性擦拭测试,以确保没有观察到污染。 放射性废物应按照当地环境健康和安全(EH& S)规定处理。

食谱

  1. 20mM Fe-NTA(1:4摩尔比) 20mM HEPES/20mM Tris缓冲液
    0.4 M NTA
    0.4M FeCl 3
    1. 制备20mM HEPES/20mM Tris缓冲液
      向450ml ddH 2 O中溶解2.38g HEPES和1.21g Tris。
      用10N HCl调节pH至6.0 用ddH 2 O使最终体积为500ml。
    2. 准备0.4 M NTA
      在20ml 20mM HEPES/20mM Tris缓冲液中,加入4.28g NTA 用10N NaOH调节pH至6.0,在开始时加入〜3ml 10N NaOH,然后逐滴加入,直至pH达到6.0(注意:pH在pH 5-7之间迅速变化)。 /> 用20mM HEPES/20mM Tris缓冲液使终体积为56ml
    3. 在100mM HCl中制备0.4M FeCl 3 为了制备100mM HCl,将500μl37%(相当于12M)HCl溶液加入到11.5ml ddH 2 O中。
      将1.08g FeCl 3·6H 2 O溶解在10ml 100mM HCl中。
    4. 制备20mM Fe-NTA(1:4摩尔比) 在内部有搅拌棒的50ml烧杯中,加入10ml 20mM HEPES/20mM Tris缓冲液 将pH电极留在溶液中,并沿着其余步骤跟踪溶液的pH 加入2ml在步骤1c中制备的0.4M FeCl 3。溶液pH值现在应为〜2 加入8ml在步骤1b中制备的0.4M NTA 用100mM NaOH调节至最终pH为6.95 用20mM HEPES/20mM Tris缓冲液使终体积为40ml 通过0.45μm过滤器单元对20mM Fe-NTA溶液进行过滤灭菌
  2. 柠檬酸铁溶液
    0.1M柠檬酸盐缓冲液
    12.5mM FeCl 3·6H 2 O 2
    1. 准备0.1 M柠檬酸盐缓冲液
      向40ml ddH 2 O中溶解2.25g Na 3 H 6 H 6 H 5 O 7,/2H·2H 2 O(柠檬酸三钠二水合物) 用10M HCl调节pH至6.6 用ddH 2 O使最终体积为50ml。
    2. 对于50ml原液,将0.169g FeCl 3·6H 2 O加入到50ml 0.1M柠檬酸盐缓冲液中。
  3. Fe标记的柠檬酸铁溶液
    0.1M柠檬酸盐缓冲液,59μLFeCl 3缓冲液 向100μl0.1M柠檬酸盐缓冲液中加入2μCi FeCl 3 子。
    混合良好。
  4. 59 Fe标记的转铁蛋白
    10mM NaHCO 3/v/v 18.75μM转铁蛋白
    20mM Fe-NTA(1:4摩尔比) 59 FeCl <3>
    1. 向50ml PBS中加入0.042g NaHCO 3。
    2. 将7.5mg载脂转铁蛋白加入到在步骤4a中制备的PBS缓冲液中的5ml NaHCO 3中。
    3. 向2.5μl20mM Fe-NTA溶液中加入2.5μl 59 FeCl 3 3。
    4. 混合在步骤4b和4c中制备的溶液。 在室温下孵育1小时以使 59 Fe结合转铁蛋白
    5. 使用Amicon TM Ultra-15离心过滤器单元除去未结合的 59 Fe。
      1. 将4ml步骤4d中制备的59 Fe标记的转铁蛋白加入Amicon TM Ultra-15离心过滤装置中。
      2. 在RT下离心5分钟×20分钟,在放射性废物容器中丢弃洗脱液。
      3. 将4ml 10mM NaHCO 3(在步骤4a中制备)加入到过滤单元中,并在室温下以5,000xg离心20分钟,弃去洗脱液。
      4. 重复步骤eiii 3次。在最后一次洗涤中,您可能希望测量洗脱液中发现的 59 Fe放射性,以确保未结合TBI的 59 Fe已被彻底清除。洗脱液 59 Fe放射性大于30 CPM可能表示需要额外的洗涤。
      5. 在过滤器单元的顶部加入4ml的10mM NaHCO 3缓冲液,并收集所有的渗余物(其含有新加入的4ml的10mM NaHCO 3和< sup-> Fe-标记的转铁蛋白,使收集体积为〜4ml)到新鲜的5ml管中。测量 Fe标记的转铁蛋白溶液的 Fe CPM,以确保足够的放射性,从而能够检测来自每只动物的组织相关的59 Fe (期望〜10,000CPM /100μl Fe-标记的转铁蛋白溶液)。储存于4°C,直到用于尾静脉注射
  5. 0.9%NaCl溶液,高压灭菌
    向每1L ddH 2 O中加入9g NaCl 高压灭菌器消毒,存储在4°C,直到使用。

致谢

这项工作得到国家卫生研究院拨款DK080706(M.D.K.)的支持。 该方案改编自Craven等人。 (1987),并且在Jenkitkasemwong等人公开了经修改的方案的简短版本。 (2015)。

参考文献

  1. Craven,CM,Alexander,J.,Eldridge,M.,Kushner,JP,Bernstein,S.and Kaplan,J。(1987)。  SLC39A14是鼠的肝细胞铁超载的发展所必需的 遗传性血色素沉着模型。细胞Metab 22(1):138-150。

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引用:Jenkitkasemwong, S., Wang, C. and Knutson, M. D. (2016). Measurement of Transferrin- and Non-transferrin-bound Iron Uptake by Mouse Tissues. Bio-protocol 6(17): e1922. DOI: 10.21769/BioProtoc.1922.
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