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Oct 2018

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Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes
人单核细胞中葡萄糖脑苷脂酶活性的流式细胞术检测   

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Abstract

Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass spectrometry-based assays, where an internal standard is used to measure the accumulation of ceramide following metabolism of the synthetic substrate C12-glucocerebroside, and the utilisation of fluorescent probes that bind active GCase and/or release fluorescent metabolites upon cleavage by GCase. Here, we describe the application of a fluorescence-activated cell sorter-based assay to efficiently quantitate GCase enzyme activity in the monocyte population of human peripheral blood mononuclear cells. The cell-permeable GCase substrate 5-(Pentafluorobenzoylamino) Fluorescein Di-beta-D-Glucopyranoside (PFB-FDGlu) provides a means to measure GCase activity, whereby enzymatic cleavage yields the green-fluorescent PFB-F dye, detectable in the FL-1 channel of a flow cytometer. An inhibitor of lysosomal GCase activity, conduritol B-epoxide, is employed to ensure specificity. This protocol provides an advantageous approach for measuring GCase activity in living individual cells.

Keywords: Glucocerebrosidase (葡萄糖脑苷脂酶), Monocyte (单核细胞), Lipid (脂质), Enzyme (酶), Flow cytometry (流式细胞计数), GBA (葡萄糖脑苷脂酶)

Background

Glucocerebrosidase (GCase), encoded by the GBA1 gene, is a lysosomal hydrolase that converts glucosylceramide to glucose and ceramide. Loss of function homozygous, and compound heterozygous missense mutations in GBA1 are associated with the accumulation of glucosylceramide and the lysosomal storage disorder Gaucher’s disease (Sidransky, 2012). Whereas heterozygous mutations in GBA1 are associated with increased risk of developing the neurodegenerative movement disorder Parkinson’s disease (PD) (Sidransky and Lopez, 2012). While GBA1 mutations occur in approximately 5-15% of PD cases (Neumann et al., 2009; Sidransky et al., 2009; Lesage et al., 2011), reduced GCase activity is also observed in PD patients without GBA1 mutations (Murphy et al., 2014; Alcalay et al., 2015; Atashrazm et al., 2018), and there is increasing evidence supporting the notion that restoring GCase function is a valid therapeutic option for PD (Schapira 2015; Sardi et al., 2018). Subsequently, a number of small molecule chaperones have been described that can restore GCase function (Khanna et al., 2010 and 2012; Patnaik et al., 2012; McNeill et al., 2014).

The assessment of GCase activity in PD patients can help inform which patients may benefit from such therapies, by identifying those with reduced enzyme activity. Assessment of GCase activity could also be used to gauge the efficacy of proposed therapies to restore GCase function. The efficacy of chaperone-based therapies for restoring GCase function have predominantly been assessed by measuring changes in GCase protein by immunoblotting, or by indirectly measuring GCase activity using mass spectrometry-based assays. Although these approaches have proven useful and do have advantages (Wolf et al., 2018), they are also limited in their ability to distinguish between blood cell types, assay GCase activity in intact living cells, and are potentially less clinically amenable than other techniques. Another current approach to measuring GCase activity involves the use of the non-natural substrate 4-MUG (4-Methylumbelliferyl β-D-galactopyranoside), which when cleaved by GCase produces the fluorescent product 4-methylumbelliferon (4-MU). However, 4-MUG is also a substrate for non-lysosomal GCase (encoded by GBA2) (Boot et al., 2007) requiring the purification of lysosomes or performing the assay at distinguishing pH to add specificity.

The current protocol presented here utilises a selective lysosomal GCase substrate 5-(Pentafluorobenzoylamino) Fluorescein Di-beta-D-Glucopyranoside (PFB-FDGlu), which is metabolised by GCase to yield fluorescein. PFB-FDGlu is cell permeable and can be used with a flow cytometer to measure GCase activity in living cells on a single-cell basis. This protocol has been adapted from earlier studies describing and validating the use of PFB-FDGlu for assessing GCase activity, primarily in the context of Gaucher’s disease (Lorincz et al., 1997; van Es et al., 1997; Rudensky et al., 2003). The ability to combine PFB-FDGlu with other phenotypic indicators, such as a viability stain and antibodies to label cell surface markers is advantageous, as it enables identification of distinct target cell populations in which GCase activity may be modulated.

Materials and Reagents

  1. Ethanol wipes (Livingstone, catalog number: LWMS )
  2. Plasters (Livingstone, catalog number: ASP23N )
  3. Needles 23G (BD Biosciences, catalog number: 367288 )
  4. BD vacutainer one-use holders (BD Biosciences, catalog number: 364815 )
  5. BD CPT sodium citrate Vacutainer Glass Cell Preparation Tubes (BD Biosciences, catalog number: 362782 )
  6. 10 μl XL Biotix Filter Tip (Interpath, catalog number: M00119FC )
  7. 100 μl Aerosol Barrier Tip (Interpath, catalog number: 24600 )
  8. 1,000 μl Aerosol Barrier Tip (Interpath, catalog number: 24800 )
  9. Falcon 5 ml round bottom polystyrene test tube with cell strainer cap, 12 x 75 mm (In Vitro Technologies, catalog number: FAL352235 )
  10. 1.5 ml EppendorfTM Safe-Lock microcentrifuge tubes (Sigma-Aldrich, catalog number: T9661 )
  11. Countess cell counting chamber slides (Life Technologies, catalog number: C10312 )
  12. Sterile syringe filter 0.22 µm (Millipore, catalog number: SLGVV255F )
  13. 1.5 ml and 15 ml centrifuge tubes
  14. PPE (lab coat, safety glasses, disposable gloves)
  15. MilliQ water
  16. Ice box and ice
  17. RPMI 1640 Medium (Thermo Fisher Scientific, catalog number: 1187093 )
  18. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: 10100147 )
  19. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2438 )
  20. Penicillin-Streptomycin (100x) (Thermo Fisher Scientific, catalog number: 15140122 )
  21. L-Glutamine (Thermo Fisher Scientific, catalog number: 25030081 )
  22. CD3 for T-lymphocytes (BD Biosciences, catalog number: 560365 )
  23. CD19 for B-lymphocytes (BD Biosciences, catalog number: 555415 )
  24. CD16 for non-classical monocytes (BD Biosciences, catalog number: 560475 )
  25. 5-(Pentafluorobenzoylamino) Fluorescein Di-beta-D-Glucopyranoside (PFB-FDGlu) (Thermo Fisher Scientific, catalog number: P11947 )
  26. PE-Cy7 Mouse Anti-Human CD14 antibody (BD Biosciences, catalog number: 557742 )
  27. PE-Cy7 Mouse IgG2a, k Isotype Control (BD Biosciences, catalog number: 557907 )
  28. 1x DPBS (Ca2+/Mg2+ free) (Thermo Fisher Scientific, catalog number: 14190144 )
  29. 1 M HEPES (Sigma-Aldrich, catalog number: H0887 )
  30. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Merck, catalog number: E5134 )
  31. Sodium Hydroxide (NaOH) (Merck, catalog number: 221465 )
  32. Trypan blue 0.4% (Thermo Fisher Scientific, catalog number: T10282 )
  33. Culture media (500 ml) (see Recipes)
  34. 0.1 M EDTA solution (500 ml) (see Recipes)
  35. Fluorescence-activated cell sorting (FACS) Buffer (500 ml) (see Recipes)
  36. CBE 50 mM stock solution (see Recipes) (Sigma-Aldrich, catalog number: C5424-5MG )
  37. PFB-FDGlu 37.5 mM stock solution (see Recipes)

Equipment

  1. Tube rack to hold 1.5, 5, 15 ml tubes
  2. Class II Biosafety cabinet
  3. Micropipettes (P10, P100, P1000)
  4. S1 Pipette Filler (Thermo Fisher Scientific, catalog number: 9521 )
  5. Heraeus Multifuge X1 Centrifuge with inserts for 15 ml conical tubes (Thermo Fisher Scientific)
  6. Centrifuge (Eppendorf, model: 5415 R )
  7. Countess II FL automated cell counter (Thermo Fisher Scientific)
  8. Hotplate magnetic stirrer
  9. Magnetic stir bar
  10. Magnetic stirring pad
  11. pH meter
  12. 4 °C refrigerator
  13. -20 °C freezer
  14. 37 °C 5% CO2 incubator
  15. Vortex
  16. Flow cytometer equipped with a 488 nm laser and 530 nm-FITC filter (i.e., BD LSR Fortessa X-20)
  17. Computer for analysis

Software

  1. BD FACSDivaTM software (BD Biosciences)
  2. FlowJo software (Tree Star, OR, USA)
  3. GraphPad Prism software (GraphPad Software Inc., CA, USA)

Procedure

  1. Aseptic technique
    Conduct experiments in a class II biological safety cabinet using appropriate PPE to maintain safety and sterility.

  2. Isolation of human peripheral blood mononuclear cells (PBMC)
    1. Perform venepuncture with the arm in a downward position. Collect venous blood into 8 ml BD Vacutainer Cell Preparation Tubes (CPT), keeping tubes upright following blood collection. Immediately prior to centrifugation, invert tubes 8 times to remix the blood sample.
    2. Centrifuge at 1,800 x g for 20 min at room temperature (21 °C) in a swing bucket centrifuge with acceleration and deceleration set at 9.
    3. Collect PBMC layer with sterile glass pipette (whitish layer just beneath the plasma) and transfer to a 15 ml tube. For more specific details and images on collecting the PBMC layer from CPT tubes please refer to the following Bio-protocol publication https://bio-protocol.org/e2103 (Puleo et al., 2017).
    4. Add room temperature 1x DPBS to bring volume to 15 ml and invert tube 5 times, followed by centrifugation for 15 min at 300 x g at room temperature.
    5. Aspirate supernatant and resuspend pellet in 10 ml of warmed (37 °C) culture media for cell counting.
    6. Dilute PBMC suspension 1:1 in Trypan blue, transfer mixture to a cell counting chamber slide, and count PBMCs using an automated cell counter.
    7. Determine the required volume to obtain 1 x 106 cells.
    Note: Cell Preparation Tubes should be centrifuged within 2 h of blood collection as GCase activity declines if blood is left longer.

  3. Distribution of PBMCs into Eppendorf tubes
    1. Add 1 x 106 PBMCs per Eppendorf tube, with a total of three Eppendorf tubes for each subject, as shown in Table 1. One tube receives CBE (tube 1) and one tube receives DMSO (tube 2), with each of these followed by the addition of PFB-FDGlu substrate. The third tube is an isotype control.

      Table 1. Distribution of PBMCs into Eppendorf tubes


  4. Addition of CBE or DMSO to appropriate tubes
    1. Centrifuge tubes at 300 x g for 5 min at room temperature and decant supernatant.
    2. Gently resuspend cells in 96 μl of culture media.
    3. Add 2 μl of 50 mM CBE stock solution into tube 1 to achieve a final concentration of 1 mM, and add 2 μl of DMSO into tube 2
    4. Vortex tubes gently to mix.
    5. Incubate at 37 °C with 5% CO2 for 60 min.

  5. Addition of PFB-FDGlu substrate to appropriate tubes
    1. Following the 60 min incubation, add 2 μl of 37.5 mM PFB-FDGlu stock solution into tubes 1 and 2 to achieve a final concentration of 0.75 mM.
    2. Vortex tubes gently to mix.
    3. Incubate at 37 °C with 5% CO2 for 30 min.
    Note: If adapting this protocol for cell types other than PBMCs it is recommended to perform time course experiments to ensure the assay remains in the linear range.

  6. Termination of reaction
    1. To terminate the reaction, add 1 ml of ice-cold FACS buffer.
    2. Pellet cells via centrifugation at 300 x g for 5 min at 4 °C and decant supernatant.

  7. Staining for CD14 positive monocytes
    1. Resuspend cells in 95 μl of ice-cold flow buffer and add 5 μl of anti-CD14 antibody into tubes 1 and 2.
    2. Add 5 μl of isotype control antibody into tube 3 if using BD. For other brands of isotype control, ensure usage at the same concentration as the BD anti-CD14 antibody.
    3. Vortex tubes gently to mix.
    4. Incubate for 20 min at 4 °C, protected from light.
    5. Add 1 ml of FACS buffer to tubes, centrifuge at 300 x g for 5 min at 4 °C, and discard supernatant.
    6. Resuspend cells in 350 μl FACS buffer.
    7. Pass each cell suspension through the cell strainer cap into the 5 ml polypropylene tube compatible with the flow cytometer. 
    8. Store tubes shielded from light at 4 °C until flow cytometry data acquisition.
    Note: Other surface marker antibodies for lymphocyte or monocyte populations can be included. Antibodies we have successfully used are CD3 for T-lymphocytes, CD19 for B-lymphocytes and CD16 for non-classical monocytes. However, fixing the cells and performing intracellular staining is detrimental to the PFB-FDGlu signal and not recommended.

  8. Data acquisition
    1. Acquire data on a BD LSR Fortessa X-20 Cell Analyser using FACSDiva or equivalent cytometer and software, collecting a minimum of 50,000 events per tube.
    2. Export FCS files from FACSDiva software.

Data analysis

  1. Gating strategy
    1. Analyse FCS files using FlowJo software. To remain objective, create the gates described below using tube 3 (isotype control tube), and then apply the gates to tubes 1 and 2 by selecting and dragging the gate series to the “All Samples” tab under the “Group” tab.
    2. Firstly, perform doublet and debris exclusion by plotting area against height for forward scatter (FSC: Figure 1A), and then for side scatter (SSC: Figure 1B), to ensure only single cells are included in the analysis. Label these “singlets FSC” and “singlets SSC”, respectively.
    3. Use FSC (size) and SSC (granularity) parameters to gate the mononuclear cell population (Figure 1C), and label this “mononuclear cells”.
    4. Next, plot CD14-PE-Cy7 fluorescence against SSC-A and select the CD14-positive population (Figure 1D), from which GCase activity will be measured. The isotype negative control tube can be used to identify and eliminate non-specific binding of the antibody, as any signal above isotype control signal can be interpreted as true CD14 positive fluorescence. Label the CD14 positive population as “CD14+ monocytes”.


      Figure 1. Representative biaxial flow cytometry plots depicting the gating strategy used to assess GCase activity in live monocytes. Events were acquired using a BD LSR Fortessa Cell Analyser. First, two singlet gates were made, using FSC (A) and SSC (B), followed by gating on PBMCs (C), and then CD14+ monocytes (D). The median fluorescence intensity resulting from PFB-FDGlu metabolism within the monocyte population was then quantified (E).

  2. Calculating GCase activity
    Express GCase activity as an index, obtained by dividing the median fluorescence intensity of cells treated without CBE by the median fluorescence intensity of cells treated with CBE. To find the median fluorescence intensity resulting from PFB-FDGlu metabolism in each tube, right-click on the “CD14+ monocytes” tab under the tube name, click “Add Statistic”, and then co-select “Median” and “FL1-A” (or equivalent channel used to detect PFB-FDGlu fluorescence). Click “Add”, and a tab displaying the median fluorescence intensity of PFB-FDGlu metabolism will appear under the “CD14+ monocytes” tab.

  3. Statistical analysis
    Perform statistical analyses using GraphPad Prism software to compare the GCase activity index between groups of interest. As an example, in our original research article (Atashrazm et al., 2018) analyses were performed to compare GCase activity between PD patients (n = 48) and healthy controls (n = 44) and this data can be found in Figure 3F.

Notes

  1. This assay may also be performed on PBMCs which have been cryopreserved. We recommend a recovery incubation period of ~2 h upon thawing, prior to beginning the assay.
  2. We have experienced large variation with different lots of PFB-FDGlu and recommend using a single lot number per experiment.
  3. We have also experienced a decline in PFB-FDGlu performance with prolonged storage after reconstitution. We recommend using reconstituted PFB-FDGlu within 1 week.

Recipes

  1. Culture media (500 ml)
    1. Combine 440 ml of RPMI 1640 with 5 ml of penicillin/streptomycin 100x, 5 ml of L-Glutamine, and 50 ml of fetal bovine serum
    2. Filter sterilize and store at 4 °C for up to one month
  2. 0.1 M EDTA solution (500 ml)
    1. Combine 18.61 g of EDTA with 500 ml MilliQ Water, using a magnetic stirring pad with heating to dissolve
    2. Adjust pH to reach 8.0 using sodium hydroxide (NaOH)
    3. Autoclave at 125 °C for 25 min to sterilize
    4. Store at room temperate for several months
  3. Fluorescence-activated cell sorting (FACS) Buffer (500 ml)
    1. Combine DPBS (Ca2+/Mg2+ free) with 1 mM EDTA, 25 mM HEPES, and 5% v/v FBS to produce FACS buffer
    2. Filter sterilize and store at 4 °C for several months
  4. CBE 50 mM stock solution
    1. Bring one 5 mg vial of CBE to room temperature and aseptically combine with 616.9 μl DMSO to achieve a stock solution of 50 mM
    2. Store as single use aliquots at -20 °C for several months
  5. PFB-FDGlu 37.5 mM stock solution
    1. Bring one 5 mg vial of PFB-FDGlu to room temperature and aseptically combine with 154 μl dimethyl sulfoxide (DMSO) to achieve a stock solution of 37.5 mM
    2. Store as single use aliquots at -20 °C for no longer than one week

Acknowledgments

This protocol is derived from a published study (Atashrazm et al., 2018) which was jointly funded by the Michael J Fox Foundation for Parkinson’s disease research and the Shake it up Australia Foundation. It is important to note that this derived protocol was itself adapted from earlier pioneering studies establishing the use of PFB-FDGlu to asses GCase activity (Lorincz et al., 1997; van Es et al., 1997; Rudensky et al., 2003).

Competing interests

The authors have no financial or non-financial competing interests to declare.

Ethics

All human blood samples were obtained with informed consent. Experiments were conducted in accordance with relevant guidelines and regulations and were approved by the University of Sydney Human Research Ethics Committee (#2016/363).

References

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简介



[摘要 ] 葡萄糖脑苷脂酶(GCase )是糖脂代谢的重要酶。GCase 酶缺乏症与人类疾病有关,GCase 活性的有效测量对于评估靶向该酶的治疗剂的功效至关重要。现有的测量GCase 活性的方法包括基于全血质谱的分析,其中使用内标测量合成底物C12-葡萄糖脑苷代谢后神经酰胺的积累,以及利用结合活性GCase 和// 的荧光探针的利用。或通过GCase 裂解后释放荧光代谢物 。在这里,我们描述了基于荧光激活细胞分选术的测定方法的应用,以有效地定量人类外周血单核细胞单核细胞群中的GCase 酶活性。细胞渗透性的GCase 衬底5-(Pentafluorobenzoylamino )荧光素二-β-D- 吡喃葡萄糖苷(PFB- FDGlu )提供了一个用于测量的GCase 活性,由此酶裂解产生的绿色荧光PFB-F染料,在FL-检测流式细胞仪的1个通道。使用溶酶体GCase 活性抑制剂,conduritol B-环氧,以确保特异性。该协议为测量活的单个细胞中的GCase 活性提供了一种有利的方法。

[背景 ] 葡萄糖脑苷脂酶(GCase ),由 GBA1基因是一种溶酶体水解酶,可将葡糖神经酰胺转化为葡萄糖和神经酰胺。GBA1 中功能纯合的丧失和复合杂合错义突变与糖基神经酰胺的积累和溶酶体贮积病高雪氏病有关(Sidransky,2012)。而GBA1 中的杂合突变与发展神经退行性运动障碍帕金森氏病(PD)的风险增加有关(Sidransky and Lopez,2012)。尽管GBA1 突变发生在大约5-15%的PD病例中(Neumann 等,2009; Sidransky 等,2009; Lesage 等,2011),但在没有GBA1 突变的PD患者中也观察到GCase 活性降低(Murphy 等人,2014;阿尔卡莱等人,2015; Atashrazm 。等人,2018) ,并且有越来越多的证据支持的概念,即恢复的GCase 功能为PD的有效治疗选择(Schapira 2015;萨尔迪。等人,2018 )。随后,已经描述了许多可以恢复GCase 功能的小分子伴侣(Khanna 等人,2010 和2012; Patnaik 等人,2012; McNeill 等人,2014)。

PD患者 中GCase 活性的评估可通过鉴定酶活性降低的患者,帮助告知哪些患者可从此类疗法中受益。GCase 活性的评估也可以用来评估提议的疗法恢复GCase 功能的功效。基于伴侣蛋白的疗法恢复GCase 功能的功效主要通过免疫印迹法测量GCase 蛋白的变化,或使用基于质谱的测定法间接测量GCase 活性来评估。尽管这些方法已被证明是有用的并且确实具有优势(Wolf 等人,2018),但它们在区分血细胞类型,测定完整活细胞中GCase 活性的能力方面也受到限制,并且与其他技术相比,临床上的适应性较差。另一电流的方法来测量的GCase 活性涉及使用非天然底物4-MUG(4-甲基伞形酮的β -D- galactopyra noside )中,w HICH当由裂解的GCase 产生荧光产物4-甲基伞形酮(4-MU)。然而,4-MUG还是非溶酶体GCase (由GBA2编码)的底物(Boot 等,2007),需要纯化溶酶体或在区分pH的条件下进行测定以增加特异性。

当前协议这里介绍利用选择性溶酶体的GCase 衬底5-(Pentafluorobenzoylamino )荧光素二-β-D- 吡喃葡萄糖苷(PFB- FDGlu ),其通过代谢的GCase 荧光素产生。PFB- FDGlu 是细胞渗透性,并且可以与流式细胞仪被用于测量的GCase 在活细胞中的单细胞基础上的活动。该方案已从早期的研究中进行了改编,这些研究描述并验证了PFB-FDGlu 用于评估GCase 活性的方法,主要是在高雪氏病的背景下(Lorincz 等,1997; van Es 等,1997; Rudensky 等, 2003)。将PFB-FDGlu 与其他表型指示剂(例如生存力染色剂和标记细胞表面标志物的抗体)组合的能力是有利的,因为它能够鉴定其中可以调节GCase 活性的不同靶细胞群。

关键字:葡萄糖脑苷脂酶, 单核细胞, 脂质, 酶, 流式细胞计数, 葡萄糖脑苷脂酶

材料和试剂


 


乙醇湿巾(Livingstone,目录号:LWMS)
抹灰(活石,目录号:ASP23N)
Needles 23G(BD Biosciences,目录号:367288)
BD真空容器一次性使用支架(BD Biosciences,目录号:364815)
BD CPT柠檬酸钠真空容器玻璃细胞制备管(BD Biosence,目录号:362782)
10 微升XL Biotix 过滤嘴(Interpath ,目录号:M00119FC)
100 微升气溶胶防护提示(Interpath ,目录号:24600)
1000 微升气溶胶防护提示(Interpath ,目录号:24800)
Falcon 5 ml圆底聚苯乙烯试管,带细胞过滤网盖,12 x 75 mm(体外技术,目录号:FAL352235)
1.5毫升Eppen 多夫TM 安全锁定微量离心管(Sigma-Aldrich公司,目录号:T9661)
Countess细胞计数室载玻片(Life Technologies,目录号:C10312)
无菌注射器过滤器0.22 µm(Millipore,目录号:SLGVV255F)
1.5 ml和15 ml离心管
PPE(实验室外套,安全眼镜,一次性手套)
MilliQ 水
冰盒和冰
RPMI 1640培养基(Thermo Fisher Scientific,目录号:1187093)
胎牛血清(FBS)(Thermo Fisher Scientific,目录号:10100147)
二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2438)
青霉素-链霉素(100x)(Thermo Fisher Scientific,目录号:15140122)
L-谷氨酰胺(Thermo Fisher Scientific,目录号:25030081)
T淋巴细胞CD3(BD Biosciences,目录号:560365 )
B淋巴细胞CD19(BD Biosciences,目录号:555415 )
非经典单核细胞CD16(BD Biosciences,目录号:560475 )
5-(Pentafluorobenzoylamino )荧光素二-β-D- 吡喃葡萄糖苷(PFB- FDGlu )(赛默飞世尔科技,产品目录号:P11947)
PE-Cy7小鼠抗人类CD14抗体(BD Biosciences,目录号:557742)
PE-Cy7小鼠IgG2a,k同种型对照(BD Biosciences,目录号:557907)
1 x DPBS(不含Ca 2+ / Mg 2 + )(Thermo Fisher Scientific,目录号:14190144)
1 M HE PES(Sigma-Aldrich,目录号:H0887)
乙二胺四乙酸二钠二水合物(EDTA)(Merck,目录号:E5134)
氢氧化钠(Merck,目录号221465)
台盼蓝0.4%(Thermo Fisher Scientific,目录号:T10282)
培养基(500毫升)(请参阅食谱)
0.1 M EDTA溶液(500 ml)(请参阅食谱)
荧光激活细胞分选(FACS )缓冲液(500 ml)(请参阅食谱)
CBE 50 mM 储备溶液(请参阅食谱)(Sigma-Aldrich,目录号:C5424-5MG)
PFB- FDGlu 37.5 毫米原液(见食谱)
 


设备


 


管架至hol 1.5、5、15 ml 试管
二级生物安全柜
微量移液器(P10,P100,P1000)
S1移液器灌装机(Thermo Fisher Scientific,目录号:9521)
带有15毫升锥形管插入件的贺利氏Multifuge X1离心机(Thermo Fisher Scientific)
离心机(Eppendor f,型号:5415 R)
Countess II FL自动细胞计数仪(Thermo Fisher Scientific)
电热板电磁搅拌器
磁力搅拌棒
磁力搅拌垫
pH计
4 °C 冰箱
-20 °C 冷冻室
37 °C 5%CO 2 培养箱
涡流
流式细胞仪配备有488nm的激光器和530nm处-FITC过滤器(即,BD LSR Fortessa X-20)
分析用电脑
 


软件


 


BD FACSDiva TM 软件(BD Biosciences)
FlowJo 软件(美国俄勒冈州Tree Star)
GraphPad Prism软件(美国加利福尼亚的GraphPad Software Inc.)
 


程序


 


                                                                                                                                            Asepti Ç 技术                                                       
使用适当的PPE在II级生物安全柜中进行实验,以保持安全性和无菌性。


 


                            人外周血单个核细胞(PBMC)的分离
手臂朝下进行静脉穿刺。将静脉血收集到8毫升的BD Vacutainer细胞制备试管(CPT)中,在采血后保持试管直立。立即离心前,转化管8次再混合所述血液样品。
在室温(21 °C )下以1800 x g的速度在离心斗式离心机中离心20分钟,加速和减速设置为9。
用无菌玻璃移液管收集PBMC层(在血浆下方为发白层)并转移至15 ml管。更具体的从CPT管收集PBMC层细节和图像请参阅下面的生物-p rotocol p ublication https://bio-protocol.org/e2103(Puleo 等人,2017)。
添加房间temperatur Ë 1X DP BS在300带来体积至15ml,并转化管5倍,然后离心15分钟X 克在室温下。
吸出上清液,然后将沉淀重悬于10 ml加热过的(37 °C )培养基中以进行细胞计数。
用台盼蓝将PBMC悬浮液1:1稀释,将混合物转移到细胞计数室玻片上,并使用自动细胞计数器对PBMC进行计数。
确定所需的体积以获得1 x 10 6个单元。
注意:细胞制备试管应在采血后2小时内离心,因为如果放血时间更长,GCase 活性会下降。


 


 


将PBMC分配到Eppendorf管中
加1×10个6 PBMC的每Eppendorf管,具有总共三个Eppendorf管为每个主题的,如图Ť 能够1.一种管接收CBE(管1)和一个管接收d MSO(管2),每个的这些之后,添加PFB-FDGlu 底物。第三管是同种型对照。
 


表1. PBMC在Eppendorf管中的分布





CBE


二甲基亚砜


PFB- FDGlu


1个


+


--


+


2


--


+


+


3


--


--


--


 


              将CBE或DMSO添加到合适的试管中
在室温下将试管以300 x g 离心5分钟,倒出上清液。
轻轻重悬细胞于96 微升培养基。
将2μl 的50 mM CBE储备溶液添加到试管1中,以达到1 mM的最终浓度,然后将2μl 的DMSO 添加到试管2中。
涡旋管轻轻混合。
在5%CO 2 和37 °C下孵育60分钟。
 


将PFB-FDGlu 底物添加到合适的试管中
在60分钟温育后,加入2 微升的37.5 毫PFB- FDGlu 原液进入管1和2,以实现0.75的终浓度毫米。
涡旋管轻轻混合。
在5%CO 2 和37 °C下孵育30分钟。
注意:如果要使该协议适用于PBMC以外的其他细胞类型,建议执行时程实验以确保测定保持在线性范围内。


 


              反应终止
要终止反应,请加入1 ml冰冷的FACS缓冲液。
通过在4 °C下以300 x g 离心5分钟沉淀沉淀细胞,并倾析上清液。
 


CD14阳性单核细胞染色
将细胞重悬在95 微升冰冷的缓冲液流动,并添加5 微升的抗CD14抗体的进入管1和2。
如果使用BD,则将5μl 同型对照抗体添加到试管3中。对于其他品牌的同型对照,请确保以与BD抗CD14抗体相同的浓度使用。
涡旋管轻轻混合。
避光放置于4 °C 孵育20分钟。
向试管中加入1 ml FACS缓冲液,在4 °C下以3 00 x g 离心5分钟,并弃去上清液。
将细胞重悬在350 微升FACS缓冲液。
将每个细胞悬液通过细胞滤网盖放入与流式细胞仪兼容的5 ml聚丙烯管中。
将试管在4 °C下避光保存,直到流式细胞仪数据采集为止。
注意:可以包括针对淋巴细胞或单核细胞群体的其他表面标记抗体。我们已成功使用的抗体是T淋巴细胞CD3,B淋巴细胞CD19 和非经典单核细胞CD16。然而,固定细胞并进行细胞内染色不利于PFB-FDGlu 信号,因此不建议使用。


 


数据采集
使用FACSDiva 或等效的细胞仪和软件在BD LSR Fortessa X-20细胞分析仪上采集数据,每管最少收集50,000个事件。
从FACSDiva 软件导出FCS文件。
 


数据分析


 


一个门控策略       


使用FlowJo 软件分析FCS文件。为了保持客观性,请使用管3(同型对照管)创建以下所述的门,然后通过选择门系列并将其拖到“组”选项卡下的“所有样品”选项卡上,将门应用于管1和2。
首先,针对前向散射(FSC:图1A),然后针对侧面散射(SSC:图1B)绘制高度与面积的关系,以进行双峰和碎片排除,以确保分析中仅包括单个单元格。分别标记这些“单一FSC”和“单一SSC”。
使用FSC(大小)和SSC(粒度)参数来控制单核细胞群(图1C),并标记此“单核细胞”。
接下来,针对SSC-A绘制CD14-PE-Cy7荧光图,并选择CD14阳性群体(图1D),从中可以测量GCase 活性。同种型阴性对照管可用于鉴定和消除抗体的非特异性结合,因为同种型对照信号之上的任何信号均可解释为真正的CD14阳性荧光。将CD14阳性群体标记为“ CD14 + 单核细胞”。
 


D:\ Reformatting \ 2020-3-2 \ 1902944--1349 Nicolas Dzamko 706808 \ Figs jpg \图1.jpg


图1.代表性的双轴流式细胞仪图描述了用于评估活单核细胞中GCase 活性的门控策略。使用BD LSR Fortessa 细胞分析仪获得事件。第一,二,单峰门是毫安日,使用FSC(甲)和SSC(乙),随后门控的PBMC(Ç ),然后CD14 + 单核细胞(d )。然后,对单核细胞群体中PFB-FDGlu 代谢产生的中值荧光强度进行定量(E )。


 


B 计算GCase 活性       


将GCase 活性表示为指标,方法是将未CBE处理的细胞的中值荧光强度除以CBE处理的细胞的中值荧光强度。要查找每个试管中PFB - FDGlu 代谢产生的中值荧光强度,请右键单击试管名称下的“ CD14 + 单核细胞”选项卡,单击“添加统计”,然后同时选择“中位数”和“ FL1- A”(或用于检测PFB-FDGlu 荧光的等效通道)。单击“添加”,一个显示PFB-FDGlu 代谢中值荧光强度的标签将出现在“ CD14 + 单核细胞”标签下。


 


C 统计分析        


使用GraphPad Prism软件执行统计分析,以比较感兴趣的组之间的GCase 活性指数。例如,在我们的原始研究文章(Atashrazm 等人,2018)中进行了分析,以比较PD患者(n = 48)和健康对照(n = 44)之间的GCase 活性,该数据可以在图3F中找到。


 


笔记


 


该测定还可以在已冷冻保存的PBMC上进行。我们建议解冻后约2小时的恢复潜伏期,然后再开始测定。
我们在不同批次的PFB-FDGlu上经历了较大的差异,建议在每个实验中使用一个批次号。
重构后,长时间存储也使PFB-FDGlu 性能下降。我们建议在1周内使用重构的PFB-FDGlu 。
 


菜谱


 


培养基(500毫升)
将440 ml RPMI 1640与5 ml青霉素/链霉素100x,5 ml L-谷氨酰胺和50 ml 胎牛血清混合
滤波器sterili ž e和储存在4 ℃下进行长达一个月
0.1 M EDTA溶液(500毫升)
使用电磁搅拌垫加热将18.61 g EDTA与500 ml MilliQ 水合并
用氢氧化钠(NaOH)调节pH值到8.0
在125 °C下高压灭菌25分钟以进行灭菌
在室温下存放几个月
荧光激活细胞分选(FACS)缓冲液(500 ml)
将DPBS(不含Ca 2+ / Mg 2+ )与1 mM EDTA,25 mM HEPES和5%v / v FBS 合并以产生FACS缓冲液
过滤器消毒的d储存在4 ℃下数月
CBE 50 mM储备溶液
带来一个5 CBE的毫克小瓶至室温,并在无菌条件下与616.9结合微升DMSO,以实现50的储备溶液毫
作为一次性使用的等分试样在-20 °C下存放几个月
PFB- FDGlu 37.5 毫米原液
带来PFB-之一5毫克小瓶FDGlu 至室温,并在无菌条件下与结合
154 微升二甲基亚砜(DMSO),以实现37.5原液毫
作为一次性使用的等分试样在-20 °C下保存不超过一周
 


致谢


 


该方案源自已发表的研究(Atashrazm et al。,2018),该研究由迈克尔·J·福克斯基金会(Michael J Fox Foundation)进行帕金森氏病研究,并与Shake it up Australia基金会共同资助。重要的是要注意,该衍生协议本身是根据早期的开创性研究改编而成的,该研究建立了使用PFB-FDGlu 评估GCase 活性的方法(Lorincz 等,1997; van Es 等,1997; Rudensky 等,2003)。。


 


 


利益争夺


 


作者没有宣布任何金融或非金融竞争利益。


 


伦理


 


所有人类血样均在知情同意下获得。实验是根据相关准则和法规进行的,并得到了悉尼大学人类研究伦理委员会的批准(#2016/363)。


 


参考文献


 


Alcalay,RN,Levy,OA,Waters,CC,Fahn,S.,Ford,B.,Kuo,SH,Mazzoni,P.,Pauciulo,MW,Nichols,WC,Gan-Or,Z.,Rouleau,GA, Chung,WK,Wolf,P.,Oliva,P.,Keutzer,J.,Marder,K. and Zhang,X.(2015年)。有和没有GBA突变的帕金森氏病中的葡萄糖脑苷脂酶活性。脑138(Pt 9):2648-2658。              
Atashrazm,F.,Hammond,D.,Perera,G.,Dobson-Stone,C.,Mueller,N.,Pickford,R.,Kim,WS,Kwok,JB,Lewis,SJG,Haldayay,GM和Dzamko, N.(2018年)。帕金森氏病患者单核细胞中的葡萄糖脑苷脂酶活性降低。科学代表8(1):15446。
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引用:Hughes, L. P., Halliday, G. M. and Dzamko, N. (2020). Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes. Bio-protocol 10(7): e3572. DOI: 10.21769/BioProtoc.3572.
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