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Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages
在原初和活化的巨噬细胞中测定刚地弓形虫的复制   

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Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune defenses; specifically, T. gondii can invade and persist within host phagocytes, using them to disseminate to the brain and central nervous system where cysts are then formed. This protocol is used to evaluate the ability of Toxoplasma gondii to survive and replicate within naive and activated murine bone marrow-derived macrophages at the level of single infected cells. In the following protocol macrophages are naive or activated with IFN-γ and LPS but different activation stimuli can be utilized as well as different host cell populations and diverse inhibitors. Parasite replication is determined by evaluating the number of parasites per vacuole over time using immunofluorescence staining for parasties and microscopic analysis. Kinetic determination of parasite number per vacuole accurately reflects parasite replication over time as vacuoles-containing parasites do not fuse with one another. Isolation of murine bone marrow-derived macrophages, preparation of conditioned L929 cells for collection of macrophage colony-stimulating factor, and staining for fluorescence microscopy included in the protocol has broad applicability. This protocol works well for pathogens like Toxoplasma gondii that reside in vacuoles that do not fuse with one another and that can be visualized by microscopy.

Keywords: Toxoplasma(弓形虫), Macrophages(巨噬细胞), Cell-autonomous immunity(细胞自主免疫), Nitric oxide(一氧化氮)

Materials and Reagent

   Note: source of reagents is not critical to experiment.

  1. Murine bone marrow derived macrophages (see protocol below)
  2. Rabbit polyclonal antibody against Toxoplasma gondii (Fitzgerald Laboratories)
  3. Alexa Fluor® 488-conjugated goat anti-rabbit IgG (H + L) (Life Technologies, Invitrogen™, catalog number: A-11012 )
  4. Dulbecco’s modified ragle medium high glucose (DMEM) (1x) (Life Technologies, Invitrogen™, catalog number: 10313-039 )
  5. Fetal calf serum (FCS) (Hyclone, catalog number: SH30396.03 ); heat inactivated 56 °C for 30 min
  6. L-gluatamine (Hyclone, catalog number: SH30034.01 )
  7. Penicillin/streptomycin (Hyclone, catalog number: SV30010 )
  8. L929 cell (ATCC CCL1) conditioned media as a source of macrophage colony-stimulating factor (M-CSF) for maturation of bone marrow-derived macrophages (see protocol below)
  9. Dulbeccos PBS (Ca2+ free and Mg2+ free) (DPBS) (Hyclone, catalog number: SH30028.02 )
  10. Toxoplasma gondii parasites (tachyzoites), Tachyzoites are the haploid replicating stage of Toxoplasma gondii that can be grown in vitro in human fibroblast cells or other host cell types.
  11. Lipopolysaccharide (LPS) (List Biological from E. Coli O55: B5 #203)
  12. Recombinant murine IFN-γ (Pepro Tech, catalog number: 50813665 )
  13. Aminoguanidine (Thermo Fisher Scientific, Acros Organics, catalog number: AC40078-1000 ) - inhibitor of inducible nitric oxide synthase
  14. Sodium nitroprusside (SNP) (Thermo Fisher Scientific, catalog number: AC211640000 ) - nitric oxide donor
  15. 16% methanol-free paraformaldehyde EM grade (Electron Microscopy Sciences, catalog number: 15710 )
  16. Triton X100 (Sigma-Aldrich, catalog number: T9284-500 )
  17. Mounting media such as Vectashield containing DAPI to stain nucleus (Vector Laboratories, catalog number: H-1200 )
  18. Cover slips for the chamber slides (22 x 50 x 1) (Thermo Fisher Scientific, catalog number: 12-548-5E )
  19. 4% paraformaldehyde
  20. KCl
  21. KH2PO4
  22. NaCl
  23. Na2HPO4
  24. DPBS (we purchase it but the recipe is below) (see Recipes)
  25. D10 media (see Recipes)
  26. BMC media (see Recipes)
  27. Blocking buffer (see Recipes)
  28. Wash buffer (see Recipes)

Equipment

  1. Fluorescence microscope with 100x phase or DIC objective
  2. Tabletop centrifuge capable of holding 15 or 50 ml centrifuge tubes
  3. Humidified CO2 incubator at 37 °C
  4. Bacteriological petri plates (100 mm x 15 mm) (Thermo Fisher Scientific, catalog number: 0875712 )
  5. Zeiss inverted Axiovert 200
  6. Hemocytometer
  7. 8-well chamber slides (BD Biosciences, catalog number: 354118 ) or cover slips inserted into wells in a 24 well plate
  8. Sterile plastic 50 ml centrifuge tubes (brand is not important)

Procedure

  1. Isolate murine bone marrow derived-macrophages (see protocol below).
  2. Grow macrophages for 1 week in BMC media (see recipe and protocol below) on bacteriological petri plates.
  3. Dislodge macrophages from bacteriological petri plates with cold DPBS (calcium and magnesium free). This is done by rinsing with cold DPBS, discarding the added DPBS, repeating the wash and then keeping the plates with PBS at 4 °C for 20 min and collecting adherent cells by rinsing monolayer with DPBS using a sterile transfer pipette or serological pipette (see detailed protocol below).
  4. Add macrophages at a concentration of 3 x 105 macrophages per ml in 250-300 μl of D10 medium to chambers at least 2 h before addition of parasites to allow macrophages to adhere. Place cover back on chamber slide and place in humidified chamber at 5% CO2 at 37 °C. Add 1 x 104 Toxoplasma gondii parasites to macrophages. Parasites can be added directly to the 250-300 μl of media already in chambers. Replace slide cover and allow parasites to invade for 4 h in a humidified incubator with 5% CO2 at a temperature of 37 °C. Generally 1-10 μl of parasites adjusted to the appropriate concentration in D10 medium is added per chamber. If you need to adjust the concentration of parasites by centrifugation followed by resuspension in fresh D10 media before adding to chambers use polystyrene or PET (polyethylene terephthalate) centrifuge tubes as parasites stick to polypropylene; centrifugation 1,700 rpm [582 rcf, 10 min].
  5. Remove the media and rinse the chamber slides with fresh D10 medium to remove any remaining extracellular parasites. In the absence of stimuli, add 250-300 μl of fresh D10 media to chamber, replace cover and incubate in humidified 5% CO2 incubator at 37 °C. Do not let the cells in the chamber slide become dry at any point in the protocol as it will kill the parasites and host cells.
  6. If applicable, activate cells by dilution of stimulating agent or inhibitors in fresh D10 medium (250-300 μl/chamber).
    1. To activate macrophages, add LPS (10 ng/ml) and/or recombinant murine IFN-γ (100 U/ml).
    2. To inhibit iNOS while activating macrophages, add LPS (10 ng/ml), recombinant murine IFN-γ (100 U/ml), and aminoguanidine (1 mM).
    3. To challenge with the NO-donor sodium nitroprusside (SNP) add 100 μM of SNP.
    4. Macrophages can also be pre-activated up to 24 h prior to parasite invasion versus after parasite invasion although the effect on parasite replication is likely to be different as macrophages activated after parasite invasion are generally more permissive for parasite growth.
  7. Incubate for 24 h in a humidified incubator 37 °C and 5% CO2. If rate of parasite proliferation or doubling time needs to be determined kinetics can be performed instead of a single end point ie 6, 12, 24 and 36 h post parasite challenge.
  8. Following desired incubation; pour off media and fix macrophage monolayers with 250 μl per chamber with 4% paraformaldehyde (diluted to 4% with 1x PBS) for 20 min at 4 °C.
  9. Rinse chambers twice with 1x DPBS. Remove DPBS. Do not let chambers dry out during staining.
  10. Add 100 μl of blocking buffer with Triton X-100 detergent (see recipe) for 30 min to block and permeabilize the host cells and parasites so that they can be reached by antibodies.
  11. While waiting, dilute the unlabelled primary antibodies to the appropriate concentrations in blocking buffer:
    1. Rabbit polyclonal antibody against Toxoplasma gondii - 1:1,000 dilution. However, concentration of antibody needs to be optimized by the user. Other antibodies against Toxoplasma gondii or directly-conjugated fluorescence antibodies can be substituted.
  12. Pour off the blocking solution and add 100 μl of the primary antibody dilution to each well. Let stand for 1 h.
  13. While waiting, dilute the fluorescently labeled secondary antibodies to the appropriate concentrations in blocking buffer:
    1. Alexa Fluor® 488 goat anti-rabbit IgG (H + L) - 1:250 dilution. Optimal concentration of antibody may need to be optimized by user.
  14. Pour off the primary antibody and rinse each well three times with wash buffer (see Recipes) for 2 min per wash. Washes are performed by dumping out solution in slide and adding wash buffer 250-300 μl per chamber and letting solution sit for approximately 2 min.
  15. Add 100 μl of the secondary antibody dilution to each well and let stand for 1 h.
  16. Pour off the secondary antibodies and rinse each well four times with wash buffer for 2 min per wash.
  17. Wash each well two times with 1x DPBS for 2 min per wash.
  18. Mount the slides with Vectashield containing DAPI or other mounting media.
  19. Observe and analyze parasites in macrophages using a fluorescent microscope with a 100x phase or differential interference contrast (DIC) objective. The internal ultrastructure of the parasite is better with phase microscopy but DIC provides a more 3-dimensional perspective. We use a Zeiss inverted Axiovert 200 motorized microscope with a 100x objective, Zeiss filter sets 31, 34, 38, and 50, and Axiovision 4.3 software.
  20. To examine the replicative rate of the parasites, a time course analysis can be performed. To examine replication the number of parasites per each PV should counted. To examine whether parasites are killed by activation the number of parasites per macrophages at 1 h compared to later time points should be examined as well. If the number of parasites per macrophages is significantly reduced over time parasite cell death has occurred. Slides can also be co-stained with lysosomal associated membrane protein-1 using a species of antibody and flourochrome different from that used to detect parasites to evaluate the extent of phagosome-lysosome fusion (Mordue and Sibley, 1997).
  21. Examine at least 100 parasite-containing vacuoles per experiment and count the number of parasites per vacuole. We suggest counting two groups of 50 vacuoles or 100 vacuoles to obtain an average plus or minus standard deviation. Note the morphology of the parasites as some activation stimuli may result in parasite death and degradation or amorphous parasite-containing vacuoles.


    Figure 1. Toxoplasma gondii replication in naive versus murine bone marrow-derived macrophages activated with IFN-γ and LPS. Parasites are shown in green and the macrophage nucleus is in blue due to the DAPI in the Vectashield mounting media. The parasites in the vacuole in naive macrophages have greater than 8 parasites/vacuole while the parasites following activation of infected macrophages have only two parasites per vacuole. Other representative pictures are shown in References 1-3.

Recipes

  1. DPBS (we purchase it but the recipe is below)
    2.7 mM KCl
    1.5 mM KH2PO4
    136.9 mM NaCl
    8.9 mM Na2HPO4 (7H2O)
  2. D10 media
    500 ml DMEM
    50 ml heat inactivated FCS (10% final concentration)
    5 ml L-glutamine (final concentration 2 mM)
    5 ml penicillin (100 U/ml final concentration) -streptomycin (100 μg/ml final concentration)
  3. BMC media
    D10 Media (recipe above)
    20% L-929 cell conditioned medium
  4. Blocking buffer
    45 ml 1x DPBS
    5 ml 10% fetal calf serum
    100 μl Triton X100 (0.2% Triton X-100)
  5. Wash buffer
    49.5 ml 1x DPBS
    0.5 ml 10% fetal calf serum (1% FCS final)

Protocol for L929 conditioned media (use sterile technique)

  1. Plate 5 x 105 L929 cells in a 150-cm2 tissue culture flask containing 50 ml of D10 medium.
  2. Grow cells to confluency in a humidified incubator with 5% CO2 at 37 °C and collect supernatant as L929 cells are beginning to round up and release from the flask. M-CSF is produced by L929 cells just prior to cell death).
  3. Collect supernatant and centrifuge it at 1,500 rpm (453 x g) (for 10 min to pellet any L929 cells remaining in the supernatant.
  4. Freeze in 40 ml aliquots in a 50 ml centrifuge tube prior to use (supernatant expands in freezer so centrifuge tube should not be too full prior to freezing).

Protocol for bone marrow isolation and bone marrow macrophage differentiation (use sterile technique)

  1. Sacrifice mouse by CO2 inhalation.
  2. Sterilize the abdomen and hind legs with 70% ethanol.
  3. Make an incision in the midline of the abdomen being careful not to penetrate the peritoneum. Pull back the skin above the peritoneum to fully expose the hind legs (both femur and tibia).
  4. Using sterile small dissecting scissors and forceps clean the femur and tibia of all muscle tissue. Separate the femur and tibia from the mouse by cutting at the hip and knee joint and knee and ankle joint and place in sterile petri dish containing 10 ml of sterile DPBS. Bones can be further cleared of muscle tissue at this point if needed.
  5. Cut at the end of both sides of the femur and tibia just enough to expose the bone marrow within.
  6. Fill a 10 ml syringe with a 25-gauge needle with 10 ml of DPBS. Insert needle into one end of the femur or tibia while holding bone firmly with forceps and flush bone marrow into a 50 ml centrifuge tube.
  7. Pipet the bone marrow cells up and down to disperse into a single cell suspension.
  8. Count the cells with a hemocytometer and adjust the concentration to 2 x 106 cells/ml in BMC media.
  9. Plate 10 ml of cells in each sterile bacteriological petri dish. The brand of bacteriological petri dish is important as macrophages adhere too tightly for easy removal on some brands and on tissue culture treated plastic.
  10. Incubate cells in a humidified incubator with 5% CO2 at 37 °C for one week. On day 5 add an additional 10 ml of BMC media to the media already in the petri dish. This is to provide fresh M-CSF but to leave in any macrophage-derived growth factor already produced by the macrophages in the petri dish.
  11. For macrophage removal remove supernatant and rinse the macrophages in the petri dish twice with DPBS without calcium and magnesium. Add 5 ml of DPBS without calcium and magnesium and place cells at 4 °C or in a refrigerator for 15-20 min. Use a sterile transfer pipette or serological pipette to dislodge macrophages off the petri plate (hold plate at slant and flush bottom of the plate. Successful dislodgement of macrophages will be visible as obvious clearance of cells from bottom of plate.
  12. Pipette macrophage-containing supernatant in 50 ml centrifuge tube and centrifuge at 1,500 rpm (453 rcf) for 10 min. Pour off supernatant and resuspend pellet in 10 ml of D10 media. Count cells using a hemocytometer and adjust to a concentration of 3 x 105 cells per ml. Add 250-300 μl to each chamber of an 8 well chamber slide. Replace cover that comes with chamber slide and Incubate cells in a humidified incubator with 5% CO2 at 37 °C overnight to let cells adhere (two hours is sufficient if cells need to be used the same day).

Acknowledgments

This work was adapted from a protocol we used for studies published in Skariah et al. (2012). The studies were funded by a grant to D.G. Mordue from the National Institute of Health (NIH) 1R01 AI 072028.

References

  1. Mordue, D. G. and Sibley, L. D. (1997). Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry. J Immunol 159(9): 4452-4459.
  2. Pollard, A. M., Skariah, S., Mordue, D. G. and Knoll, L. J. (2009). A transmembrane domain-containing surface protein from Toxoplasma gondii augments replication in activated immune cells and establishment of a chronic infection. Infect Immun 77(9): 3731-3739.
  3. Skariah, S., Bednarczyk, R. B., McIntyre, M. K., Taylor, G. A. and Mordue, D. G. (2012). Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates. J Immunol 188(7): 3404-3415.

简介

弓形虫是一种专性的细胞内原生动物寄生虫,其引起疾病弓形体病。慢性感染通过主要在心脏和神经组织中形成组织囊肿来建立。 T的定义特性。 gondii 是其逃避宿主免疫防御的能力;具体地,em。 gondii可以侵入并持续在宿主吞噬细胞内,使用它们传播到脑和中枢神经系统,然后形成囊肿。该方案用于评估弓形虫在单个感染细胞水平下在幼稚和活化的鼠骨髓衍生的巨噬细胞中存活和复制的能力。在以下方案中,巨噬细胞是幼稚的或用IFN-γ和LPS活化的,但是可以利用不同的激活刺激以及不同的宿主细胞群体和不同的抑制剂。寄生虫复制通过使用针对寄生虫和显微镜分析的免疫荧光染色评估每个液泡随时间的寄生虫数量来确定。每个液泡的寄生虫数的动力学测定准确地反映了随时间的寄生虫复制,因为含有空泡的寄生虫不彼此融合。分离鼠骨髓来源的巨噬细胞,制备用于收集巨噬细胞集落刺激因子的条件性L929细胞,并且包括在方案中的荧光显微镜染色具有广泛的适用性。该协议对于存在于不彼此融合并且可通过显微镜可视化的液泡中的病原体如弓形体病毒(inxoplasma gondii)起作用。

关键字:弓形虫, 巨噬细胞, 细胞自主免疫, 一氧化氮

材料和试剂

   注意:试剂来源对实验并不重要。

  1. 鼠骨髓来源的巨噬细胞(见下面的协议)
  2. 针对弓形虫的兔多克隆抗体(Fitzgerald Laboratories)
  3. Alexa Fluor 488-缀合的山羊抗兔IgG(H + L)(Life Technologies,Invitrogen TM,目录号:A-11012)
  4. Dulbecco氏改良氏培养基高葡萄糖(DMEM)(1x)(Life Technologies,Invitrogen TM,目录号:10313-039)
  5. 胎牛血清(FCS)(Hyclone,目录号:SH30396.03); 热灭活56℃30分钟
  6. L-岩藻胺(Hyclone,目录号:SH30034.01)
  7. 青霉素/链霉素(Hyclone,目录号:SV30010)
  8. L929细胞(ATCC CCL1)条件培养基作为巨噬细胞集落刺激因子(M-CSF)的来源,用于骨髓来源的巨噬细胞的成熟(参见下面的方案)
  9. Dulbeccos PBS(Ca 2+和游离的Mg 2+)(DPBS)(Hyclone,目录号:SH30028.02)
  10. 弓形体寄生虫(tachyzoite),速殖子是弓形体的单倍体复制阶段,其可以在人成纤维细胞或其他宿主细胞类型中体外生长。
  11. 脂多糖(LPS)(来自大肠杆菌O55:B5#203的List Biological)
  12. 重组鼠IFN-γ(Pepro Tech,目录号:50813665)
  13. 氨基胍(Thermo Fisher Scientific,Acros Organics,目录号:AC40078-1000) - 诱导型一氧化氮合酶抑制剂
  14. 硝普钠(SNP)(Thermo Fisher Scientific,目录号:AC211640000) - 一氧化氮供体
  15. 16%无甲醇的多聚甲醛EM级(Electron Microscopy Sciences,目录号:15710)
  16. Triton X100(Sigma-Aldrich,目录号:T9284-500)
  17. 安装介质如含有DAPI的Vectashield染色核(Vector Laboratories,目录号:H-1200)
  18. 室玻片(22×50×1)(Thermo Fisher Scientific,目录号:12-548-5E)的盖玻片
  19. 4%多聚甲醛
  20. KCl
  21. KH 2 PO 4
  22. NaCl
  23. Na HPO 4
  24. DPBS(我们购买,但食谱在下面)(见配方)
  25. D10媒体(见配方)
  26. BMC媒体(见配方)
  27. 阻止缓冲区(参见配方)
  28. 洗涤缓冲液(见配方)

设备

  1. 具有100x相或DIC物镜的荧光显微镜
  2. 能够容纳15或50 ml离心管的台式离心机
  3. 在37℃下加湿CO 2培养箱
  4. 细菌培养皿(100mm×15mm)(Thermo Fisher Scientific,目录号:0875712)
  5. Zeiss逆转Axiovert 200
  6. 血细胞计数器
  7. 8孔室玻片(BD Biosciences,目录号:354118)或插入24孔板的孔中的盖玻片
  8. 无菌塑料50ml离心管(品牌不重要)

程序

  1. 分离鼠骨髓衍生的巨噬细胞(参见下面的方案)
  2. 在细菌培养皿上在BMC培养基中培养巨噬细胞1周(参见下面的配方和方案)
  3. 从具有冷DPBS(不含钙和镁)的细菌培养皿中取出巨噬细胞。这通过用冷DPBS冲洗,弃去添加的DPBS,重复洗涤,然后将板用PBS在4℃保持20分钟,并通过使用无菌转移移液管或血清移液管用DPBS洗涤单层来收集贴壁细胞来进行详细协议如下)。
  4. 在添加寄生虫以允许巨噬细胞粘附之前,将在250-300μlD10培养基中的浓度为3×10 5个巨噬细胞/ml的巨噬细胞加入室至少2小时。将盖放回室载玻片上,并置于37℃,5%CO 2的潮湿室中。向巨噬细胞中添加1 x 10 4 弓形虫寄生虫。寄生虫可以直接添加到已经在腔室中的250-300μl的培养基中。更换滑盖,并允许寄生虫在37℃的温度下在具有5%CO 2的湿润培养箱中侵入4小时。通常在每个室中加入在D10培养基中调节至适当浓度的1-10μl寄生虫。如果需要通过离心来调节寄生虫的浓度,然后在添加到室中之前在新鲜D10培养基中重悬,使用聚苯乙烯或PET(聚对苯二甲酸乙二醇酯)离心管作为寄生虫粘附到聚丙烯;离心1,700rpm [582rcf,10min]
  5. 取出介质,用新鲜的D10培养基冲洗腔玻片,以去除任何残留的细胞外寄生虫。在没有刺激的情况下,向室中加入250-300μl新鲜的D10培养基,替换盖并在37℃下在湿润的5%CO 2培养箱中孵育。不要让室玻片中的细胞在方案中的任何点变干,因为它会杀死寄生虫和宿主细胞。
  6. 如果适用,通过在新鲜D10培养基(250-300μl/室)中稀释刺激剂或抑制剂来活化细胞。
    1. 为了激活巨噬细胞,加入LPS(10ng/ml)和/或重组鼠IFN-γ(100U/ml)。
    2. 为了在激活巨噬细胞时抑制iNOS,加入LPS(10ng/ml),重组鼠IFN-γ(100U/ml)和氨基胍(1mM)。
    3. 用NO-供体硝普钠(SNP)攻击加入100μMSNP
    4. 巨噬细胞也可以在寄生虫侵入之前相对于寄生虫侵入后预活化24小时,虽然对寄生虫复制的影响可能不同,因为寄生虫侵入后激活的巨噬细胞通常更容许寄生虫生长。
  7. 在37℃和5%CO 2的潮湿培养箱中孵育24小时。 如果需要确定寄生虫增殖或倍增时间的速率,可以进行动力学,而不是单个终点,即寄生虫攻击后6,12,24和36小时。
  8. 在所需温育后;倾倒培养基并用4%多聚甲醛(用1×PBS稀释至4%)在4℃下每孔用250μl固定巨噬细胞单层20分钟。
  9. 用1x DPBS冲洗两次。删除DPBS。在染色过程中不要让室干燥。
  10. 加入100微升含Triton X-100洗涤剂(参见配方)的封闭缓冲液30分钟,以阻断和透化宿主细胞和寄生虫,以便它们可以被抗体到达。
  11. 在等待时,将未标记的一级抗体在封闭缓冲液中稀释至适当的浓度:
    1. 兔多克隆抗体对弓形虫 - 1:1,000稀释。然而,抗体的浓度需要由用户优化。针对弓形虫的其他抗体或直接偶联的荧光抗体可以被替代。
  12. 倒掉封闭溶液,向每个孔中加入100μl一抗稀释液。让1小时。
  13. 在等待时,将荧光标记的第二抗体在封闭缓冲液中稀释至适当的浓度:
    1. Alexa Fluor 488山羊抗兔IgG(H + L)-1:250稀释。 抗体的最佳浓度可能需要由用户优化。
  14. 倒出一抗,并用洗涤缓冲液(参见Recipes)将每个孔漂洗3次,每次洗涤2分钟。 通过将溶液倾倒在载玻片上并加入每个室250-300μl的洗涤缓冲液并使溶液静置约2分钟进行洗涤。
  15. 向每个孔中加入100μl次级抗体稀释液,放置1小时
  16. 倒出第二抗体并用洗涤缓冲液冲洗每个孔四次,每次洗涤2分钟
  17. 用1x DPBS洗涤每个孔两次,每次洗涤2分钟
  18. 使用包含DAPI或其他安装介质的Vectashield安装幻灯片。
  19. 使用具有100x相位或微分干涉对比(DIC)目标的荧光显微镜观察和分析巨噬细胞中的寄生虫。寄生虫的内部超微结构更好与相显微镜,但DIC提供了更多的三维透视。我们使用具有100倍物镜的Zeiss倒置Axiovert 200电动显微镜,Zeiss滤光片组31,34,38和50以及Axiovision 4.3软件。
  20. 为了检查寄生虫的复制率,可以进行时间过程分析。为了检查复制,应该计数每个PV的寄生虫数量。为了检查寄生虫是否通过激活而被杀死,与之后的时间点相比,在1小时时每个巨噬细胞的寄生虫数目也应该被检查。如果每个巨噬细胞的寄生虫数量随着时间的推移显着降低,则发生寄生虫细胞死亡。载玻片还可以使用与用于检测寄生虫的抗体和荧光染料不同的物种与溶酶体相关的膜蛋白-1共染色,以评价吞噬体 - 溶酶体融合的程度(Mordue和Sibley,1997)。
  21. 每个实验检查至少100个含寄生虫的液泡,并计算每个液泡的寄生虫数量。我们建议计数两组50个液泡或100个液泡以获得平均加或减标准偏差。注意寄生虫的形态,因为一些激活刺激可能导致寄生虫死亡和退化或无定形寄生虫空泡。


    图1.在用IFN-γ和LPS激活的原始骨髓来源的巨噬细胞中的原始弓形虫复制。寄生虫显示为绿色,巨噬细胞核为蓝色 到Vectashield封装介质中的DAPI。 初始巨噬细胞中液泡中的寄生虫具有大于8个寄生虫/液泡,而在感染的巨噬细胞活化后的寄生虫每个液泡只有两个寄生虫。 其他代表性图片如参考文献1-3所示

食谱

  1. DPBS(我们购买,但食谱在下面)
    2.7 mM KCl
    1.5mM KH 2 PO 4 4/v/v 136.9mM NaCl 8.9mM Na 2 HPO 4(7H 2 O)。
  2. D10媒体
    500 ml DMEM
    50ml热灭活的FCS(10%终浓度) 5ml L-谷氨酰胺(终浓度2mM) 5 ml青霉素(100 U/ml   链霉素(100μg/ml终浓度)
  3. BMC媒体
    D10媒体(上述食谱)
    20%L-929细胞条件培养基
  4. 阻塞缓冲区
    45 ml 1x DPBS
    5ml 10%胎牛血清 100μlTriton X100(0.2%Triton X-100)
  5. 洗涤缓冲液
    49.5 ml 1x DPBS
    0.5ml 10%胎牛血清(最终1%FCS)

L929条件培养基的方案(使用无菌技术)

  1. 在含有50ml D10培养基的150-cm 2组织培养瓶中平板接种5×10 5个L929细胞。
  2. 在37℃下在具有5%CO 2的湿润培养箱中培养细胞至融合,并收集上清液,因为L929细胞开始圆形化并从烧瓶中释放。 M-CSF由L929细胞在细胞死亡之前产生)
  3. 收集上清液,并以1,500rpm(453×g)离心(10分钟,沉淀上清液中剩余的任何L929细胞。)
  4. 使用前在50 ml离心管中冷冻40 ml等分试样(上清液在冷冻箱中扩增,这样离心管在冷冻前不应过满)。

骨髓分离和骨髓巨噬细胞分化方案(使用无菌技术)

  1. 通过CO 2吸入杀死小鼠。
  2. 用70%乙醇消毒腹部和后腿。
  3. 在腹部的中线做一个切口,小心不要穿透腹膜。拉回腹膜上方的皮肤以完全暴露后腿(股骨和胫骨)。
  4. 使用无菌小型解剖剪刀和镊子清洁所有肌肉组织的股骨和胫骨。通过在髋关节和膝关节和膝关节和踝关节切割,将股骨和胫骨与小鼠分开,并置于含有10ml无菌DPBS的无菌培养皿中。如果需要,骨骼可以在这一点进一步清除肌肉组织
  5. 切割在股骨和胫骨两侧的末端,足以暴露骨髓。
  6. 使用25号针头用10ml DPBS填充10ml注射器。将针头插入股骨或胫骨的一端,同时用镊子牢牢固定骨头,并将骨髓冲入50 ml离心管中。
  7. 吸取骨髓细胞上下分散成单细胞悬液
  8. 用血细胞计数器计数细胞,并在BMC培养基中将浓度调节至2×10 6个细胞/ml。
  9. 板10毫升细胞在每个无菌细菌培养皿。细菌培养皿的品牌是重要的,因为巨噬细胞粘着得太紧,以致于不容易在某些品牌和组织培养处理的塑料上移除。
  10. 将细胞在具有5%CO 2的潮湿培养箱中在37℃下孵育1周。在第5天,向已经在培养皿中的培养基中再加入10ml BMC培养基。这是为了提供新鲜的M-CSF,但留下 已经由培养皿中的巨噬细胞产生的任何巨噬细胞衍生的生长因子
  11. 对于巨噬细胞去除,去除上清液,并用不含钙和镁的DPBS冲洗培养皿中的巨噬细胞两次。加入5ml无钙和镁的DPBS,将细胞置于4℃或冰箱中15-20分钟。使用无菌转移移液管或血清移液管将巨噬细胞从培养皿上取下(将培养板倾斜并冲洗在培养板底部)。从培养皿底部清除细胞时,巨噬细胞成功移位。
  12. 吸移含巨噬细胞的上清液在50ml离心管中,并在1,500 rpm(453 rcf)离心10分钟。倒出上清液并将沉淀重悬于10ml D10培养基中。使用血细胞计数器计数细胞并调节至3×10 5个细胞/ml的浓度。向8孔腔室载玻片的每个室中加入250-300μl。更换与腔室玻片一起的盖子,并在37℃下,在具有5%CO 2的潮湿培养箱中孵育细胞过夜,以使细胞粘附(如果细胞需要在同一天使用,则两小时就足够了) 。

致谢

这项工作改编自我们用于在Skariah等人(2012)中公开的研究的方案。研究资金由D.G.来自国家健康研究所(NIH)1R01 AI 072028的Mordue。

参考文献

  1. Mordue,D.G。和Sibley,L.D。(1997)。 在形成时确定包含弓形虫的液泡的胞内命运,并取决于进入的机制。
  2. Pollard,A.M.,Skariah,S.,Mordue,D.G.and Knoll,L.J。(2009)。 A  来自弓形虫的含有跨膜结构域的表面蛋白 增强活化免疫细胞中的复制和建立 慢性感染。 Infect Immun 77(9):3731-3739。
  3. Skariah,S.,Bednarczyk,R.B.,McIntyre,M.K.,Taylor,G.A.and Mordue,D.G。(2012)。 发现对寄生虫对反应性氮中间体有抗性的重要的新型弓形体锥体相关蛋白。 a> J Immunol 188(7):3404-3415
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Iaconetti, E., Lynch, B., Kim, N. and Mordue, D. G. (2012). Determination of Toxoplasma gondii Replication in Naïve and Activated Macrophages. Bio-protocol 2(22): e289. DOI: 10.21769/BioProtoc.289.
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