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Dec 2017
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Isolation of GFP-expressing Malarial Hypnozoites by Flow Cytometry Cell Sorting
流式细胞术分离表达GFP的疟原虫催眠素   

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Abstract

Hypnozoites are dormant liver-stage parasites unique to relapsing malarial species, including the important human pathogen Plasmodium vivax, and pose a barrier to the elimination of malaria. Little is known regarding the biology of these stages, largely due to their inaccessible location. Hypnozoites can be cultured in vitro but these cultures always consist of a mixture of hepatocytes, developing forms, and hypnozoites. Here, using a GFP-expressing line of the hypnozoite model parasite Plasmodium cynomolgi, we describe a protocol for the FACS-based isolation of malarial hypnozoites. The purified hypnozoites can be used for a range of ‘-omics’ studies to dissect the biology of this cryptic stage of the malarial life cycle.

Keywords: Malaria (疟疾), Plasmodium cynomolgi (猴疟原虫), Plasmodium vivax (间日疟原虫), Hypnozoites (休眠子), Relapse (复发), FACS (流式细胞术细胞分选), Cell sorting (细胞分选)

Background

Many gaps in knowledge surround hypnozoite biology, which hampers drug discovery efforts. This is largely due to the inaccessible location of hypnozoites inside the liver in vivo and their presence in low numbers (in contrast to the size of the liver, which contains an estimated 3.6 × 1011 cells) (Bianconi et al., 2013.)


In vitro liver-stage cultures have been developed but the percentage of infected hepatocytes is at maximum 3% (Zeeman et al., 2014), which complicates ‘-omics’ studies aiming to characterize hypnozoites in more detail. The host cell genome (Yan et al., 2011) is 100-fold larger in size than the parasite genome (Pasini et al., 2017), indicating that hepatocyte-derived material will be a major source of contamination for ‘-omics’ studies of hypnozoites. Moreover, in vitro cultures (Sattabongkot et al., 2006; Dembele et al., 2011; Gural et al., 2018; Roth et al., 2018) contain a mixture of uninfected hepatocytes and liver-stage schizonts and hypnozoites; therefore, not only is parasite enrichment needed but also separation of hypnozoites from the developing forms.


For several malarial species, including P. yoelii, P. berghei, and P. falciparum, methods have been described to purify liver stages by FACS using fluorescent reporter lines (Natarajan et al., 2001; Tarun et al., 2006; Prudencio et al., 2008); however, these species do not develop into hypnozoites. Only a few primate malarial parasite species form hypnozoites.


P. vivax liver-stage research is dependent on patient material, which complicates experimentation; nonetheless, RNAseq of P. vivax hypnozoites has been performed (Gural et al., 2018) through selective drug treatment of liver-stage cultures, precluding a direct comparison between hypnozoites and liver-stage schizonts.


The closely phylogenetically related monkey parasite, P. cynomolgi, is more accessible to experimentation and has been used extensively as a model for P. vivax (Joyner et al., 2016; Zeeman et al., 2016; Gupta et al., 2019). In fact, hypnozoites were first identified in P. cynomolgi (Krotoski et al., 1983) and extensive drug studies involving P. cynomolgi have shown very similar responses between P. vivax and P. cynomolgi (Schmidt, 1983). While working with monkeys is ethically restricted and limited to labs that have access to non-human primates, the P. cynomolgi parasite model offers unique possibilities for screening compounds for hypnozoiticidal activity and to gain a better understanding of hypnozoite biology. A previous study used laser-capture dissection of P. cynomolgi hypnozoites for transcriptomics (Cubi et al., 2017), which enables small-scale sampling of fixed parasites. To allow sampling of larger quantities of (live) hypnozoites, here, we describe a method for the FACS sorting of hypnozoites.


P. cynomolgi is accessible to genetic engineering (Kocken et al., 1999) and this has enabled the development of fluorescent reporter lines for FACS (Voorberg-van der Wel et al., 2013; Voorberg-van der Wel, 2020), permitting the large-scale isolation of live malarial hypnozoites and liver-stage schizonts as described in this protocol (Voorberg-van der Wel et al., 2017; Bertschi et al., 2018).


Materials and Reagents

  1. Collagen-coated 96-well plates (PerkinElmer, catalog number: 6055700)

  2. Cellstar 50 ml polypropylene tubes (Greiner Bio-One, catalog number: 227261)

  3. 5 ml serological pipettes (for example, VWR, catalog number: 612-5523)

  4. 10 ml serological pipettes (for example, Greiner Bio-One, catalog number: 607-107)

  5. 25 ml serological pipettes (for example, VWR, catalog number: 612-5544)

  6. 1.5 ml Sarstedt tubes, sterile (Sarstedt, catalog number: 72.692.405)

  7. 5 ml round-bottomed polystyrene test tubes (Falcon, catalog number: 352052)

  8. 5 ml round-bottomed polystyrene test tubes, with cell strainer snap cap (Falcon, catalog number: 352235)

  9. Reagent reservoir (Greiner Bio-One, catalog number: 960305)

  10. Pipette tips 300 µl and 1,000 µl (Rainin, SR LTS filter tip 768)

  11. Phosphate-buffered saline (PBS, 1×) (Thermo Fisher Scientific, catalog number: 14200-067)

  12. 0.25% trypsin/EDTA (Thermo Fisher Scientific, catalog number: 25200072)

  13. TRIzol reagent (Thermo Fisher Scientific, catalog number: 15596026)

  14. William’s E medium + Glutamax (Thermo Fisher Scientific, catalog number: 32551-087)

  15. Human serum A+ (pooled, heat inactivated; Sanquin blood bank)

  16. Insulin/transferrin/selenium supplement 100× (Thermo Fisher Scientific, catalog number: 41400-045)

  17. Sodium pyruvate 100 mM (Thermo Fisher Scientific, catalog number: 11360-036)

  18. MEM-NEAA 100× (Thermo Fisher Scientific, catalog number: 11140-035)

  19. Pen/strep 100× (Thermo Fisher Scientific, catalog number: 15140-122)

  20. Hydrocortisone (Sigma, catalog number: H0888)

  21. 50 mM β-mercaptoethanol (Thermo Fisher Scientific, catalog number: 31350-010)

  22. DMSO hybrimax (Sigma, catalog number: P1860)

  23. William’s B medium (see Recipes)

  24. 20% William’s B medium (see Recipes)

  25. 0.1 M hydrocortisone (see Recipes)


Primary cells and parasites

  1. Primary macacque hepatocytes (freshly isolated or thawed from a cryopreserved stock; Macaca mulatta or Macaca fascicularis; source: BPRC; hepatocytes may be obtained through commercial suppliers

  2. GFP-expressing P. cynomolgi lines as described in Voorberg-van der Wel et al. (2013 and 2020); lines can be obtained upon request from the corresponding author

Equipment

  1. (Multichannel) pipettes (Rainin)

  2. Pipette controller (Integra Biosciences, model: Pipetboy acu 2)

  3. Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra X-15R)

  4. Inverted microscope (Leica, model: DMi1)

  5. Laminar flow biosafety cabinet IIB (Clean-Air EF/B6)

  6. Fume hood

  7. Humidified 5% CO2 incubator at 37°C

  8. Water bath at 37°C

  9. Ice machine

  10. Vortex

  11. High Content Imager – Operetta (PerkinElmer)

  12. Cell sorter – FACS Aria I flow cytometer (BD Biosciences) equipped with a blue laser (20 mW)

  13. -80°C freezer (Eppendorf)

Software

  1. FlowJo software version 9.9 (https://www.flowjo.com/solutions/flowjo/downloads/previous-versions)

  2. FACS Diva version 8.0.1 (https://www.bdbiosciences.com/en-us/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)

  3. Columbus 2.8.2 (PerkinElmer) (https://www.perkinelmer.com/nl/product/image-data-storage-and-analysis-system-columbus)

Procedure

The different steps of the protocol are depicted in Figure 1 and described in detail below.



Figure 1. Overview of the procedure. Briefly, primary macaque hepatocytes are plated into collagen-coated 96-well plates, and after 2-3 days are inoculated with GFP-expressing P. cynomolgi sporozoites isolated from A. stephensi mosquitoes. At 6 days post-inoculation the culture is imaged using an Operetta high content imager to assess the presence of hypnozoites and schizonts. The cells are then detached by trypsinization and this pooled material is used for flow cytometry cell sorting of hypnozoites and schizonts.


  1. Infection and culture of macaque primary hepatocytes with transgenic P. cynomolgi parasites

    1. Plate 65 × 103 macaque primary hepatocytes per well (96-well plates; see Note 2) and maintain the culture at 5% CO2 and 37°C in William’s B medium (100 µl per well; see Recipe 1) supplemented with 2% DMSO. Hepatocytes can be freshly isolated or used from a cryopreserved stock. After 2-3 days, inoculate the cultures with 50 × 103 fluorescent transgenic P. cynomolgi sporozoites per well (Voorberg-van der Wel et al., 2013 ) as described in https://bio-protocol.org/e3722.

    2. Maintain the culture in William’s B medium without DMSO (this affects parasite development) at 5% CO2 and 37°C; refresh the medium three times per week by removing as much culture medium as possible, followed by gently adding fresh William’s B medium (100 µl per well). Routinely, the culture is maintained for 6 days because at that time, hypnozoites and developing liver stages can be distinguished based on their size.

    3. Culture alongside uninfected wells with macaque primary hepatocytes as the control (≥6 wells of a 96-well plate).


  2. Harvesting cells

    1. On the day of harvest, check the quality of infection (for reference) by live imaging on a fluorescence microscope/High Content Imager (Voorberg-van der Wel et al., 2013 ). Please see the image in the flowchart for an example. Further insights into liver-stage parasite development can be gained from Voorberg-van der Wel et al. (2020) .

    2. Remove the supernatant from the 96-well plates and wash with 1× in PBS (100 µl/well).

    3. Remove the supernatant and add 30-50 µl pre-warmed (at 37°C) 0.25% trypsin/EDTA per well; incubate for 3 min at 37°C until the cells have detached (check with an inverted microscope).

    4. Add 100 µl William’s B medium per well to stop trypsinization.

    5. Mix by gently pipetting up and down (using a multichannel pipette) and pool the contents of the wells (containing the detached cells and fluids) into a reagent reservoir.

    6. Transfer the material into a 50-ml tube and spin (2 min 200 × g, RT, brake medium).

    7. Wash twice in 20% William’s B medium (each time spin 2 min 200 × g, RT, brake medium).

    8. Pass the sample, while gently pipetting up and down, through a cell strainer snap cap (on top of 5-ml round-bottomed polystyrene test tubes) to exclude clumps.

    9. Keep cells on ice until sorting.


  3. Fluorescence-activated cell sorting

    1. Pre-cool the sample loader at 4°C and rotate at 300 rpm during cell sorting.

    2. Set up the FACSAria according to the manufacturer’s procedures, e.g., run Cytometer Setup and Tracking beads (CS&T).

    3. Install a 100-µm nozzle and set the sheath pressure to 20 psi (default setting).

    4. Record 100,000 events of the uninfected hepatocyte culture as well as the transgenic liver-stage parasite culture to check the gating settings.

    5. Insert 1.5-ml collection tubes filled with 300 µl TRIzol if the material is to be used for transcriptomics

      NB: TRIzol is toxic: fill the tubes in the fume hood and dispose of the waste separately.

    6. Sort (sort precision: 4-way purity mode) the fractions of interest (Figure 2).



      Figure 2. Gating strategy for sorting hypnozoites and schizonts on day 6. Parasite populations are gated based on FITC expression. The GFPlow gate contains hypnozoites and the GFPhigh gate contains developing forms (schizonts). The GFPdim gate contains a mixture of parasites.


    7. When approximately half of the sample has been sorted, sort a small amount of sample into 100 µl 20% William’s B (collect about 100 GFPlow and GFPhigh events each; also take a sample of the other fractions at the same time) and transfer to a 96-well plate for a quality check of the Operetta system (Voorberg-van der Wel et al., 2013 ).

    8. When the sort has finished, vortex the TRIzol tubes for 30 s and transfer to the -80°C freezer.

Notes

  1. Please note that working with the materials listed above potentially involves biological risks. Monkey-derived hepatocytes should be treated as biohazard material and may contain infectious agents. P. cynomolgi parasites are infectious to humans. All handling of these materials should be performed under BSL2 conditions.

  2. While the original work was performed in 6-well plates, we have noticed that infection grades in 96-well plates are higher.

  3. Instead of using uninfected hepatocytes as the negative control, macaque primary hepatocytes infected with P. cynomolgi wild-type parasites may be used (Voorberg-van der Wel et al., 2013 ).

  4. Hepatocytes are highly autofluorescent; therefore, it is difficult to distinguish between uninfected cells and those containing GFPlow-expressing parasites (hypnozoites). We have empirically determined that using the PE channel in combination with the FITC channel provides an efficient method to separate the different parasitic populations.

  5. Implementing a doublet and debris exclusion through the addition of a scatterplot of FSC versus SSC, followed by a scatterplot of FSC-A versus FSC-H, may be beneficial for removing subcellular debris and multi-cell aggregates.

  6. Instead of harvesting all the wells, it is recommended to perform live imaging and fix a few wells for IFA (https://bio-protocol.org/e3722) to establish the infection characteristics.

  7. Co-staining with Sytox Blue can be included as a live/dead indicator, as described by others ( Posfai et al., 2018 ). Alternatively, a surface stain found on all intact hepatocytes could potentially be used in combination with, for example, PE-Cy7.

  8. Depending on the cell sorting apparatus, a 130-µm nozzle can be used as an alternative to a 100-µm nozzle. This may be beneficial for maintaining host cell integrity.

Recipes

  1. William’s B medium

    William’s E medium with glutamax

    10% human serum (A+)

    1% MEM nonessential amino acids (NEAA)

    2% penicillin/streptomycin

    1% insulin/transferrin/selenium

    1% sodium pyruvate

    50 µM β-mercaptoethanol

    0.05 µM hydrocortisone


    1. 500 ml William’s E medium

    2. 50 ml Human Serum A+

    3. 5 ml 100× pen/strep (10,000 U/ml)

    4. 5 ml 100× insulin/transferin/selenium supplement

    5. 5 ml 100 mM sodium pyruvate

    6. 5 ml 100× MEM-NEAA

    7. 250 µl 0.1 M hydrocortisone

    8. 500 µl 50 mM β-mercaptoethanol

    9. Store at 4°C and use within 1 month

  2. 20% William’s B medium

    Dilute William’s B medium 1:5 with William’s E medium

  3. 0.1 M hydrocortisone

    1. Dissolve 50 mg hydrocortisone in 1 ml DMSO

    2. Store 250-µl aliquots at 4°C

Acknowledgments

This work was supported by a grant from the Medicines for Malaria Venture, a translational research grant (WT078285) from the Wellcome Trust, FP7 EU grants MALSIG (contract number 223044) and EVIMALAR (contract number 242095), and the Bill and Melinda Gates foundation (OPP1141292).

Ethics

Nonhuman primates were used because no other models (in vitro or in vivo) were suitable for the aims of this project. The local independent ethics committee conforming to Dutch law (BPRC Dier Experimenten Commissie, DEC) approved the research protocol (agreement number DEC# 708) prior to the beginning of the study, and the experiments were performed according to Dutch and European laws. Further details can be found in the original publication (Voorberg-van der Wel et al., 2017).

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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简介

[摘要] Hypnozoites都处于休眠状态的肝脏-独特的阶段寄生虫对复发疟疾升的物种,其中包括重要的人类病原体间日疟原虫,并造成障碍消除疟疾。关于这些阶段的生物学知之甚少,主要是由于它们的位置不便。Hypnozoites可以培养在体外,但是这些培养物总是由肝细胞,显影形式的混合物,和hypnozoites。在这里,使用次生孢子模型寄生虫的食蟹疟原虫的GFP表达系,我们描述了一个协议,为FACS -疟疾的基于隔离升hypnozoites。纯化的次生孢子可用于一系列“-组学”研究,以剖析疟疾生命周期这一隐秘阶段的生物学特性。


[背景]中号的任何差距在知识环绕hypnozoite生物学,这阻碍了药物发现工作。这主要是由于肝脏里hypnozoites的难以接近的位置的体内以及它们在低的数字(存在于肝脏,其中包含的估计3.6的大小对比度× 10 11细胞)(比安科尼等人,2013年)。

体外肝脏-舞台文化已经开发,但感染的肝细胞的百分率为最多3% (塞曼等,2014) ,其复杂化“组学”研究,旨在更详细地描述hypnozoites。宿主细胞基因组(Yan等人,2011)的大小是寄生虫基因组(Pasini等人,2017)的100倍,表明肝细胞衍生的物质将是“ -组学”的主要污染源。次生动物的研究。此外,体外培养(Sattabongkot等,2006 ; Dembele等,2011; Gural等,2018; 罗斯等。,2018)包含未感染的肝细胞的混合物和肝-阶段裂殖和hypnozoites ; 因此,不仅是必要的寄生虫丰富而且从hypnozoites分离的发展形式。

对于几个疟疾升物种,包括P.疟原虫,P.疟原虫,和疟原虫,方法已被描述来纯化肝阶段通过FACS使用荧光报告线(纳塔拉詹等人,2001;塔伦等人,2006 ;普鲁登西奥等人,2008);^ h H但是,这些品种不会发展成hypnozoites。只有少数灵长类动物疟疾升寄生虫种类形成hypnozoites。

间日疟原虫肝-阶段研究依赖于患者的材料,其中complicat ES实验; Ñ onetheless,RNA测序的间日疟原虫hypnozoites已被执行(Gural等人,通过选择性药物治疗肝2018)-阶段培养,排除hypnozoites和肝脏之间的直接比较-阶段裂殖。

的密切系统发育相关的猴疟原虫,P.食蟹猴,是对实验更容易获得,并已被广泛地用作一个用于模型间日疟原虫(乔伊纳等人,2016;塞曼等人,2016;古普塔。等人,2019)。事实上,hypnozoites首先在确定P.食蟹猴(Krotoski等,1983) ,并在广泛的药物研究volving P.食蟹显示之间非常相似的反应间日疟原虫和P.食蟹猴(施密特,1983 )。虽然与猴子工作的道德限制,仅限于有机会获得非人类灵长类动物实验室的P.食蟹猴疟原虫模型提供了筛选化合物的独特的可能性hypnozoiticidal活动,以更好地hypnozoite生物学的理解。的先前的研究中使用激光捕获解剖P.食蟹猴hypnozoites为创见ř iptomics(Cubi酒店等人,2017) ,这使得能够固定寄生虫小规模取样。为了让大批量的(活)hypnozoites的采样,在这里,我们描述了一个方法的FACS hypnozoites进行排序。

P.食蟹是基因工程的访问(Kocken等,1999) ,这已使荧光报告线为FACS发展(Voorberg -范德WEL等,2013; Voorberg,范德WEL,2020年),permitt荷兰国际集团的活米的大型隔离alaria升hypnozoites和肝-如在本协议中所述阶段裂殖(Voorberg-范德瓦WEL等人,2017 ; Bertschi等人,2018) 。

关键字:疟疾, 猴疟原虫, 间日疟原虫, 休眠子, 复发, 流式细胞术细胞分选, 细胞分选




材料和试剂
胶原-包被的96 -孔板(珀金埃尔默,目录号:6055700)
Cellstar 50 ml聚丙烯管(Greiner Bio-One ,目录号:227261)
5 ml血清移液器(例如,VWR ,目录号:612-5523)
10毫升血清移液管(例如,格雷纳生物ö NE ,目录号:607-107)
25毫升血清移液管(例如,VWR ,目录号:612-5544)
1.5 ml无菌Sarstedt管(Sarstedt ,目录号:72.692.405)
5毫升ř ound -b ottom编p olystyrene吨EST吨ubes中(Falcon ,目录号:352052)
5毫升ř ound -b ottom编p olystyrene吨EST吨ubes,与Ç ELL小号训练小号打盹Ç AP中(Falcon ,目录号:352235)
试剂容器(Greiner Bio-One ,目录号:960305)
移液器吸头300 µl和1,000 µl(Rainin ,SR LTS过滤器吸头768)
磷酸盐缓冲盐水(PBS,1 × )(Thermo Fisher Scientific ,目录号:14200-067)
0.25%t胰蛋白酶/ EDTA(Thermo Fisher Scientific ,目录号:25200072)
Ť RI ZOL ř eagent(赛默飞世尔科技,产品目录号:15596026)
威廉姆斯E介质+ Glutamax (Thermo Fisher Scientific ,目录号:32551-087)
人类小号erum A +(汇集,热灭活; Sanquin血库)
胰岛素/转铁蛋白/硒补充剂100 × (Thermo Fisher Scientific ,目录号:41400-045)
丙酮酸钠100 mM(Thermo Fisher Scientific ,目录号:11360-036)
MEM-NEAA 100 × (Thermo Fisher Scientific ,目录号:11140-035)
笔/链球菌100 × (Thermo Fisher Scientific ,目录号:15140-122)
氢化可的松(Sigma ,目录号:H0888)
的50mMβ -米ercap toethanol (赛默飞世尔科技,产品目录号:31350-010)
DMSO hybrimax (Sigma ,目录号:P1860)
威廉姆斯(William's)B培养基(请参阅食谱)
20%威廉姆斯(William's)B培养基(请参阅食谱)
0.1 M h氢化可的松(请参阅食谱)


原代细胞和寄生虫

初级macacque肝细胞(新鲜分离或从冷冻保存的备料中解冻;猕猴或猕猴蟹猴;源:BPRC;肝细胞可通过商业供应商获得
如Voorberg-van der Wel等人所述,表达GFP的食蟹猕猴系。(2013和2020); 可以根据通讯作者的要求获得


设备



(多声道)p ipettes(的Rainin )
移液管Ç ontroller(的Integra Biosciences公司,型号:Pipetboy ACU 2)
冷藏b enchtop离心机(Beckman Coulter公司,型号:Allegra的X-15R)
倒置显微镜(Leica ,型号:DMi1)
层流生物安全柜IIB(Clean-Air EF / B6)
通风柜
37°C时加湿的5%CO 2培养箱
37°C水浴
制冰机
涡流
高内涵成像仪– Operetta(PerkinElmer)
细胞分选仪–装有蓝色激光(20 mW )的FACS Aria I流式细胞仪(B D Biosciences )
-80°C冰柜(Eppendorf)


软件



FlowJo软件9.9版(https://www.flowjo.com/solutions/flowjo/downloads/previous-versions)
FACS Diva版本8.0.1(https://www.bdbiosciences.com/zh-cn/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)
哥伦布2.8.2(PerkinElmer)(https://www.perkinelmer.com/nl/product/image-data-storage-and-analysis-system-columbus)


程序



该协议的不同步骤在图1中进行了描述,并在下面进行了详细描述。






图1.过程概述。简单地说,主猕猴肝细胞铺板于胶原-包被的96孔板中,并在2-3天后用表达GFP的接种P.食蟹猴从子孢子中分离A.按蚊蚊子。接种后6天,使用Operetta高含量成像仪对培养物成像以评估次生子和裂殖体的存在。所述细胞随后通过胰蛋白酶消化分离并收集该材料用于流动cytometr ÿ细胞hypnozoites和裂殖体排序。



感染和猕猴原代肝细胞的培养物与转基因P.食蟹猴寄生虫
板65 × 10 3个猕猴原代肝细胞每孔(96孔板;参见Ñ OTE 2)和维持培养在5%CO 2和37℃下在威廉B培养基(每孔100μl;参见ř ecipe 1)补充含2%DMSO。可以从冷冻保存的原液中新鲜分离或使用肝细胞。后2-3 d AYS,接种培养物以50 × 10 3荧光转基因P.食蟹猴sporozoit ES每孔(Voorberg-范德瓦WEL等人,2013年)中所描述https://bio-protocol.org/e3722。
在5%CO 2和37°C下,在无DMSO的情况下保持William's B培养基的文化(这会影响寄生虫的发育);每周去除三次培养基,每周三次刷新培养基,然后轻轻加入新鲜的William B培养基(每孔100 µl)。按常规,将培养物维持6天,因为在那个时候,hypnozoites和发展阶段的肝脏可以根据它们的大小来区分。
培养用并排猕猴原代肝细胞未感染孔的控制(≥6孔96的-孔板)。


收获细胞
在收获的当天,通过在荧光显微镜/高含量成像仪上进行实时成像检查感染的质量(以供参考)(Voorberg-van der Wel等人,2013)。请参见流程图中的图像作为示例。进一步深入了解肝-阶段寄生虫的发展可以从获得Voorberg -van德WEL等。(2020)。
除去的从96孔板中的上清液和洗瓦特我第1 × PBS中的(1 00微升/孔)。
除去的上清液,加入30-50 μL预温(37℃)0.25%吨rypsin / EDTA每孔; 孵育为在37℃下3分钟,直至该细胞分离(用倒置显微镜检查)。
每孔加入100 µl William's B培养基以停止胰蛋白酶消化。
通过轻轻地上下吹打混匀(我们荷兰国际集团的多通道pipett e)和凝聚孔的内容物(包含分离的细胞和液体)到AR eagent水库。
材料转移到50 -毫升管和自旋(2分钟200 ×克,RT,制动介质)。
在20%William's B培养基中洗涤两次(每次旋转2分钟200 × g ,RT,制动培养基)。
通过该样品,同时轻轻上下吹吸,通过细胞过滤器卡扣盖(5顶部-毫升园-底编聚苯乙烯试管)排除团块。
将细胞放在冰上直到分类。


荧光激活细胞分选
预冷却的在4℃下样品装载器和期间的细胞分选以300rpm旋转。
设置了FACSAria根据该厂家专业URE ř的程序,例如,运行流式细胞仪设置和跟踪珠(CS&T) 。
安装一个100 -微米的喷嘴并设置在鞘压力至20psi的(缺省设置)。
记录的100,000个事件的未感染的肝细胞,以及在转基因的肝脏-阶段寄生虫文化,以检查该水道荷兰国际集团设置。
插入1.5 -填充用300μlml收集管Ť RI ZOL如果所述材料将用于转录
NB :Ť RI ZOL是有毒:填充所述管在通风橱和处置的所述分开废物。

排序(排序精度:4-way纯度模式)感兴趣的馏分(图2)。





图2.在第6天排序次生子和裂殖藻的选通策略。寄生虫种群是基于FITC表达进行门控的。GFPlow门包含次生子,GFPhigh门包含发育形式(裂殖体)。所述GFPdim栅极包含寄生虫的混合物。



当pproximately样品的一半已排序,排序样品少量入100微升20%威廉的乙(收集大约100个GFPlow和GFP高每个事件;还采取的样品的其它组分在相同的时间),并转移对于质量检查○96孔板˚F的歌剧系统(Voorberg-范德瓦WEL等人,2013年)。
完成分类后,将T RI zol管涡旋30 s,然后转移到-80°C冰箱中。


笔记



请注意,使用上面列出的材料可能会涉及生物风险。猴-衍生的肝细胞应该被视为生物危害材料,并且可包含传染剂。P.食蟹猴寄生虫是感染人类。所有处理的这些材料应当BSL2条件下进行。
虽然最初的工作是在6孔板中进行的,但我们注意到96孔板中的感染等级更高。
代替使用未感染的肝细胞作为所述阴性对照,感染了猕猴原代肝细胞P.食蟹猴的野生型寄生虫可以使用(Voorberg-范德瓦WEL等人,2013年)。
肝细胞高度自体荧光; 吨herefore,很难区分之间未感染的细胞以及那些含GFPlow -表达寄生虫(hypnozoites)。我们已经凭经验确定,使用的PE通道中COMBIN通货膨胀与FITC通道提供一个Ñ高效的方法来分离不同的parasit IC种群。
实施通过双峰和碎屑的排除的阿迪和灰FSC相对于SSC的散点图,随后FSC-A对FSC-H的散点图,可以是用于去除亚细胞碎片和多细胞聚集体是有益的。
相反,收获所有的井,建议进行活体成像和修复的IFA(几井https://bio-protocol.org/e3722)建立感染特性。
共染色用SYTOX蓝可以被包括作为一个活/死指示器,如由其他人所描述(Posfai等人,2018) 。可替换地,表面污点上的所有完整的肝细胞中发现可能与组合使用,例如,PE-Cy7的。
根据排序细胞荷兰国际集团装置,130 -微米的喷嘴可以被用作一个替代100 -微米的喷嘴。这可能是有益的保持荷兰国际集团宿主细胞的完整性。


菜谱



威廉姆斯B媒介
William的E培养基,最大谷量

10%人血清(A +)

1%MEM非必需氨基酸(NEAA)

2%青霉素/链霉素

1%胰岛素/转铁蛋白/硒

1%丙酮酸钠

50 µMβ-巯基乙醇

0.05 µM氢化可的松



500毫升William E培养基
50毫升人血清A +
5毫升100 × p en / strep(10,000 U / ml)
5 ml的100 ×我nsulin /转铁蛋白/硒补充
5 ml 100 mM丙酮酸钠
5毫升100 × MEM-NEAA
250 µl 0.1 M氢化可的松
500 µl 50 mMβ-巯基乙醇
储存在4°C并在1个月内使用
20%William's B中等
用威廉姆斯E培养基稀释威廉姆斯的B培养基1:5

0.1 M h氢化可的松
一种。溶解50毫克ħ ydrocortisone 1 ml的DMSO     

b。在4°C下储存250 - µl等分试样     



致谢



这项工作是由来自疟疾新药研发公司,从一个转化型研究资助(WT078285)的资助威康信托,FP7欧盟授予MALSIG(合同编号223044),并EVIMALAR(合同号242095) ,以及比尔和梅林达·盖茨基金会(OPP1141292)。



伦理



使用非人类的灵长类动物是因为没有其他模型(体外或体内)适合该项目的目标。当地独立伦理小号委员会符合荷兰国际集团到荷兰法律(BPRC底儿Experimenten Commissie ,DEC)批准了研究协议(协议号DEC #708)之前的研究开始,并进行实验,根据荷兰和欧洲的法律。可以在原始出版物中找到更多详细信息(Voorberg-van der Wel等人,2017)。



参考



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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Voorberg-van der Wel, A., Hofman, S. O. and Kocken, C. H. M. (2021). Isolation of GFP-expressing Malarial Hypnozoites by Flow Cytometry Cell Sorting. Bio-protocol 11(9): e4006. DOI: 10.21769/BioProtoc.4006.
  2. Voorberg-van der Wel, A., Roma, G., Gupta, D. K., Schuierer, S., Nigsch, F., Carbone, W., Zeeman, A. M., Lee, B. H., Hofman, S. O., Faber, B. W., Knehr, J., Pasini, E., Kinzel, B., Bifani, P., Bonamy, G. M. C., Bouwmeester, T., Kocken, C. H. M. and Diagana, T. T. (2017). A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite Plasmodium cynomolgi.Elife 6: e29605.
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