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Macrophage Survival Assay Using High Content Microscopy
使用高内涵显微技术进行巨噬细胞的生存分析   

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Abstract

Macrophages maintain tissue homoeostasis by regulating inflammation and tissue repair mechanisms. Thus, the fate of macrophages has an impact on the state of the tissue. The aim of this protocol is to quantify macrophage survival using high content microscopy and image processing software. Here, we describe a high-content image based protocol to assess the effect of diverse stimuli in combination with pharmacological treatments on macrophage survival in a quantitative, unbiased and high-throughput manner.

Keywords: Macrophage(巨噬细胞), Cell survival(细胞存活), High-content microscope(高内涵显微镜)

Background

Macrophages are phagocytic innate immune cells and are the main drivers of inflammation in tissue (Medzhitov, 2008). These cells are associated with cancer together with autoimmune, autoinflammatory, infectious, neurodegenerative and metabolic diseases (Ginhoux and Jung, 2014). In this context, the role of macrophages in inflammation is well-studied, however, the impact of macrophage survival in non-infectious and infectious diseases is largely unknown. Our study showed that the activation of certain pathogen-associated receptors (PRRs) can induce macrophage survival (Eren et al., 2016). We described a molecular mechanism that demonstrated how an obligate intracellular pathogen exploits PRR-induced cell survival (Eren et al., 2016). Thus, further studies are necessary to understand the role of macrophage survival in different disease settings.

Materials and Reagents

  1. Sterile 1.5 ml tubes (Corning, Axygen®, catalog number: MCT-175-C )
  2. 25 G-needle
  3. 50 ml syringe (B. Braun Medical, catalog number: 4617509F-02 )
  4. Polypropylene conical 50 ml centrifuge tube (TPP Techno Plastic Products, catalog number: 91050 )
  5. 40 µM cell strainer (Corning, Falcon®, catalog number: 431750 )
  6. 90 mm Petri dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101RTC )
  7. 96-well clear bottom cell-culture grade black imaging plates (Corning, Falcon®, catalog number: 353219 )
  8. Sterile reagent reservoir (VWR, catalog number: 89094-664 )
  9. 10 ml serological pipette (SARSTEDT, catalog number: 86.1254.001 )
  10. 25 ml serological pipette (SARSTEDT, catalog number: 86.1685.001 )
  11. 10 μl filtered barrier tip (Biotix, Neptune®, catalog number: BT10XL )
  12. 200 μl filtered tip low retention (CLEARLINE, catalog number: 713117 )
  13. 0.22 μm syringe-filter (Carl Roth, catalog number: P668.1 )
  14. Adhesive plate seal
  15. 6-to-9 week old specific-pathogen free C57BL/6 mice
  16. ddH2O
  17. Ethanol
  18. Macrophage colony stimulating factor (M-CSF) (ImmunoTools, catalog number: 12343115 )
  19. EDTA 0.5 M pH 8.0 solution (as described Reference 1)
  20. Pharmalogical inhibitor
  21. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  22. HEPES buffer (BioConcept, catalog number: 5-31F00-H )
  23. Penicillin-streptomycin (P/S) (BioConcept, catalog number: 4-01F00-H )
  24. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
  25. Sodium hydroxide (NaOH)
  26. Hydrochloric acid (HCl)
  27. Paraformaldhyde (PFA) (Sigma-Aldrich, catalog number: 76240 )
    Note: This product has been discontinued.
  28. Cell-culture grade Ca/Mg-free Dulbecco’s PBS (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14040091 )
  29. Saponin (Sigma-Aldrich, catalog number: 84510 )
  30. DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
  31. Phalloidin (Thermo Fisher Scientific, InvitrogenTM, catalog number: A12379 )
  32. Complete DMEM cell medium (see Recipes)
  33. 4% PFA solution (see Recipes)
  34. 5% Saponin solution (see Recipes)
  35. Staining solution (see Recipes)

Equipment

  1. 10-11 cm long stainless steel dissecting scissors
  2. 10-11 cm long stainless steel dissecting straight forceps
  3. Refrigerator centrifuge with 50 ml tube adapter
  4. Cell culture incubator
  5. Pipette controller
  6. Pipettes
  7. Cell counting chamber
  8. Finnpipette® 50-300 μl 12 channel multi-pipette
  9. Chemical hood
  10. Luminal flow hood
  11. Plate washer (BioTek Instruments, model: EL406 )
  12. High content microscope (such as Molecular Devices, model: ImageXpress Micro XL )
  13. 40x Plan Apo λ 0.95 NA objective (Nikon, catalog number: MRD00405 )
  14. pH meter

Procedure

  1. Preparation of murine bone-marrow derived macrophages
    1. Euthanize mice using carbon-dioxide. Keep scissors and forceps in 70% ethanol during the procedure. Make a small incision on the central dorsum. Peel the skin from the top of each hind leg and down over the foot. Cut off the hind legs at the hip bone with scissors. Carefully cut off the foot and hip bones without compromising the integrity of the tibia and femur. Separate the tibia and femur by severing at the joint.
    2. Remove excess muscle from the tibia and femur. Hold bones with forceps and spray with 70% ethanol.
    3. Place bones in sterile tubes containing ice-cold DMEM with P/S. Keep tubes on ice.
    4. Using forceps, soak the bones in 70% ethanol for a few seconds. Wait for the excess ethanol to drip off before cutting each extremity of the bone with scissors to expose the bone marrow cavity.
    5. Flush the bone marrow out, using a 25 G-needle attached to a 50 ml syringe containing 25 ml of ice-cold complete DMEM medium (see Recipes), into a sterile 50-ml canonical centrifuge tube. Filter the sample through a 40 μm cell strainer into a fresh 50-ml tube on ice.
      Note: All liquid steps must be performed using sterile and pyrogen-free material under a laminal flow hood following good cell culture practice to avoid the activation of macrophages.
    6. Centrifuge cells at 450 x g for 8 min at 4 °C.
    7. Discard the supernatant by inverting the tube in one single move. Resuspend cells in ice-cold complete DMEM containing 50 ng/ml M-CSF.

  2. Culturing macrophages
    1. Count cells and adjust cell number to ~7 x 106/ml in complete DMEM containing M-CSF. Add ~7 x 105 cells per Petri dish in a final volume of 10 ml complete DMEM containing M-CSF. Incubate Petri dishes in a 37 °C, 5% CO2 incubator.
    2. On day 3, add 5 ml of fresh pre-warmed (37 °C) complete DMEM containing 50 ng/ml M-CSF to each plate.

  3. Harvest macrophages
    1. On day 6, discard the culture supernatants. Immediately add 10 ml of ice-cold cell-culture grade sterile 1x PBS without calcium and magnesium, pH 7.4 containing 5 mM EDTA. Incubate Petri dishes ~3 min on ice.
    2. Hold the Petri dish at a 30 °C angle. Flush the Petri dish with its contents using a pipette controller and a sterile pipette. Transfer the detached cells to a 50 ml sterile tube containing one-sixth of the transferred volume of complete DMEM.
    3. Centrifuge the tube at 200 x g for 8 min at 4 °C.
    4. Count cells using a cell counting chamber or an automated cell counter. Adjust the cell concentration to 1.25 x 106 cells/ml.

  4. Plating macrophages
    Pipette 100 μl of cells into 96-well clear bottom black plates using a multi-channel pipette or an automated cell dispenser under a laminar flow hood (Figure 1). Incubate cells overnight in an incubator (37 °C, 5% CO2) to ensure attachment of cells.
    Note: Cells should be dispensed on the edge of wells to have homogenous distribution. Shaking or swirling of the plates must be avoided to prevent the accumulation of cells in certain areas of the wells (Figure 2).


    Figure 1. Aspiration and dispension of liquids should be performed on the edge of the plate. Hold the plate at an approximately 30° angle to the surface and hold the multipipette at a 120° angle to the plate when aspirating or dispensing liquid into the wells to avoid cell detachment.


    Figure 2. An example of 7 x 7 tile images from wells. A and B. Show a heterogeneous cell distribution that can be caused by cell plating, plate shaking/swirling, and/or aspiration/dispension. C. Shows a homogenous cell distribution. Images display DAPI-stained fluorescent nuclei of macrophages.

  5. Treatment of macrophages
    1. Cells can be pre-treated or treated with a pharmacological inhibitor at an optimized concentration and for an optimized duration. In the case of pre-treatment, wash the wells with pre-warmed 37 °C complete DMEM, and add 100 μl of complete DMEM.
      Notes:
      1. Aspiration and dispension steps of media must be performed on the edge of wells to avoid detaching the cells (Figure 2).
      2. Following this protocol, perform a two-fold serial-dilution of a selective pharmacological inhibitor to determine non-toxic concentrations for primary macrophages. Test the potency of the pharmacological inhibitor at determined concentrations in the time course of the experiment using appropriate biochemical and/or cellular experimental approaches. We can recommend to use 1 μM staurosporine (negative control) to induce macrophage death and 10-50 ng/ml M-CSF (positive control) to induce macrophage survival and proliferation.
    2. Treat the cells with a stimuli of interest in 100 μl complete DMEM. Incubate cells at 37 °C, 5% CO2.
      Note: The duration of incubation needs to be optimized for each individual experimental set-up. It should be noted that there is a basal reduction in macrophage number after 48 h of incubation in cell culture-media.

  6. Fixation, staining, and washing steps
    1. Aspirate the cell supernatant using a multi-channel pipette or an automated plate washer.
    2. Dispense 50 μl of 4% PFA (see Recipes) in 1x PBS, pH 7.4 and incubate the plates at room-temperature for 10 min.
    3. Wash the wells twice with 200 μl 1x PBS using a multi-channel pipette or an automated plate washer.
    4. Aspirate the supernatant, and dispense 50 μl staining solution. Incubate the plate with the staining solution (see Recipes) in a dark place for 10 min.
    5. Wash the wells twice with 200 μl 1x PBS using a multi-channel pipette or an automated plate washer.
    6. Seal plates with an adhesive plate seal either manually or using an automated plate sealer.

  7. Image acquisition
    1. Clean the bottom of each 96-well plate with 70% ethanol.
    2. Acquire 49 images per well that consist of 7 x 7 images, 200 μm apart from each other, using a high content microscope equipped with a 40x objective.

  8. Counting cells with nuclear and F-actin staining
    1. Cell scoring object function of Metaexpress was used to segment and define all nuclei (DAPI channel). As a positive marker, cytoplasm staining (phalloidin channel) was used (Figure 3A). Approximate minimum and maximum width was set at 5 μm and 30 μm for ‘all nuclei’ and ‘positive marker’ parameters (Figure 3B).
      Note: Image analysis can be performed using ImageJ, Icy or Cell-profiler that are freeware image-analysis programs.
    2. A logical operation that merges nuclei and cytoplasmic objects was performed to define a cell as an object in the algorithm (Figure 3B).
    3. Cells that had cytoplasm bordering the image were removed from the image to count cells only in the field of view (Figure 3B).
    4. The cell count was taken as output data from the image analysis pipeline.


      Figure 3. The image processing and object segmentation pipeline. A. Unprocessed DAPI, phalloidin, and composite image; B. Object segmentation of nuclei and cytoplasm is shown. A cell is defined by merging nuclei and cytoplasm binary images. Bordering cells were removed in the final processed image that were used to quantify cell number.

Data analysis

Each independent experiment should be performed with three technical or biological replicates. In order to pool the independent experiments, the data can be normalized to the cell count in untreated condition or to the cell count in vehicle-treated condition if a pharmacological inhibitor was used. A bar graph and the appropriate statistical test can be used to represent the data.

Recipes

  1. Complete DMEM cell medium
    50 ml FBS
    5 ml HEPES
    5 ml P/S
    500 ml DMEM
    Filter FBS, HEPES and P/S into DMEM using a 0.22 μm filter and a 50 ml syringe
  2. 4% PFA solution
    45 ml ddH2O
    1 N NaOH
    1 N HCl
    5 ml 10x PBS
    2 g PFA
    Heat ddH2O to 60 °C. Add 2 g PFA into the tube while it is stirring under a chemical hood. Add a few drops of 1 N NaOH. Wait until the solution becomes clear. Add 10x PBS and adjust the pH to 7.4 using a pH meter and 1 N HCl. Filter the solution using a 0.22 μm and a 50 ml syringe. Aliquot and store at -20 °C
  3. 5% Saponin solution
    50 ml 1x DPBS
    2.5 g Saponin
    Dissolve Saponin by stirring. Filter the solution using a 0.22 μm and a 50 ml syringe. Aliquot and store at -20 °C
  4. Staining solution
    5 μg/ml DAPI (nucleic acid stain) (stock concentration 5 mg/ml)
    1 U/ml Phalloidin (F-actin stain) (stock concentration 200 U/ml)
    0.05% (w/v) Saponin (stock concentration 5%, w/v)
    1x DPBS, pH 7.4

Acknowledgments

We thank Dimitri Monreau and Cansel Ustunel Eren for assistance with the high-content microscopy and Slavica Masina for critical reading of the manuscript. We thank the NCCR Geneva Access platform for providing the equipment for the high-content microscopic experiments. This work was funded by grants from the Swiss National fund for research (FNRS 310030-153204 and IZRJZ3_164176, N.F.), the Institute for Arthritis Research (iAR), and the COST action (CM1307 SEFRI: C14.0070, N.F.). This protocol was adapted from the protocol described in Eren et al. (2016).

References

  1. EDTA. (2006). Cold Spring Harbor Protoc pdb.rec8030.
  2. Eren, R. O., Reverte, M., Rossi, M., Hartley, M. A., Castiglioni, P., Prevel, F., Martin, R., Desponds, C., Lye, L. F., Drexler, S. K., Reith, W., Beverley, S. M., Ronet, C. and Fasel, N. (2016). Mammalian innate immune response to a Leishmania-resident RNA virus increases macrophage survival to promote parasite persistence. Cell Host Microbe 20(3): 318-328.
  3. Ginhoux, F. and Jung, S. (2014). Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol 14(6): 392-404.
  4. Medzhitov, R. (2008). Origin and physiological roles of inflammation. Nature 454(7203): 428-435.

简介

巨噬细胞通过调节炎症和组织修复机制来维持组织均匀平衡。 因此,巨噬细胞的命运对组织的状态有影响。 该方案的目的是使用高含量显微镜和图像处理软件来量化巨噬细胞存活。 在这里,我们描述了一种基于高含量图像的方案,以定量,无偏差和高通量方式评估不同刺激与药理学治疗对巨噬细胞存活的影响。
【背景】巨噬细胞是吞噬先天免疫细胞,是组织炎症的主要驱动因素(Medzhitov,2008)。 这些细胞与自身免疫,自身炎症,感染性,神经变性和代谢性疾病一起与癌症相关(Ginhoux和Jung,2014)。 在这种情况下,巨噬细胞在炎症中的作用得到了很好的研究,然而,非传染性和感染性疾病中巨噬细胞存活的影响在很大程度上是未知的。 我们的研究表明,某些病原体相关受体(PRR)的激活可以诱导巨噬细胞存活(Eren等,2016)。 我们描述了一种分子机制,证明专门的细胞内病原体如何利用PRR诱导的细胞存活(Eren等,2016)。 因此,需要进一步的研究来了解巨噬细胞存活在不同疾病环境中的作用。

关键字:巨噬细胞, 细胞存活, 高内涵显微镜

材料和试剂

  1. 无菌1.5ml管(Corning,Axygen ,目录号:MCT-175-C)
  2. 25 G针
  3. 50ml注射器(B.Braun Medical,目录号:4617509F-02)
  4. 聚丙烯锥形50ml离心管(TPP Techno Plastic Products,目录号:91050)
  5. 40μM电池过滤器(Corning,Falcon ®,目录号:431750)
  6. 90 mm培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:101RTC)
  7. 96孔清除的底部细胞培养级黑色成像板(Corning,Falcon ®,目录号:353219)
  8. 无菌试剂库(VWR,目录号:89094-664)
  9. 10ml血清移液管(SARSTEDT,目录号:86.1254.001)
  10. 25 ml血清移液管(SARSTEDT,目录号:86.1685.001)
  11. 10μl过滤屏障尖端(Biotix,Neptune ®,目录号:BT10XL)
  12. 200μl过滤提示低保留(CLEARLINE,目录号:713117)
  13. 0.22μm注射器过滤器(Carl Roth,目录号:P668.1)
  14. 胶版印章
  15. 6至9周龄特异性病原体免费C57BL / 6小鼠
  16. ddH 2 O
  17. 乙醇
  18. 巨噬细胞集落刺激因子(M-CSF)(ImmunoTools,目录号:12343115)
  19. EDTA 0.5M pH 8.0溶液(如参考文献1所述)
  20. 药用抑制剂
  21. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  22. HEPES缓冲液(BioConcept,目录号:5-31F00-H)
  23. 青霉素 - 链霉素(P / S)(BioConcept,目录号:4-01F00-H)
  24. Dulbecco改良的Eagle's培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:31966021)
  25. 氢氧化钠(NaOH)
  26. 盐酸(HCl)
  27. 对甲醛(PFA)(Sigma-Aldrich,目录号:76240)
    注意:本产品已停产。
  28. 细胞培养级Ca / Mg的Dulbecco's PBS(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14040091)
  29. 皂苷(Sigma-Aldrich,目录号:84510)
  30. DAPI(Thermo Fisher Scientific,Invitrogen TM,目录号:D1306)
  31. 鬼笔环肽(Thermo Fisher Scientific,Invitrogen TM,目录号:A12379)
  32. 完成DMEM细胞培养基(参见食谱)
  33. 4%PFA溶液(见配方)
  34. 5%皂苷溶液(见配方)
  35. 染色溶液(参见食谱)

设备

  1. 10-11厘米长不锈钢解剖剪刀
  2. 10-11厘米长不锈钢解剖直镊
  3. 冰箱离心机带50ml管接头
  4. 细胞培养箱
  5. 移液器控制器
  6. 移液器
  7. 细胞计数室
  8. Finnipipette ® 50-300μl12通道多吸管器
  9. 化学罩
  10. 流明罩
  11. 洗板机(BioTek Instruments,型号:EL406)
  12. 高含量显微镜(如Molecular Devices,型号:ImageXpress Micro XL)
  13. 40x计划Apoλ0.95 NA目标(尼康,目录号:MRD00405)
  14. pH计

程序

  1. 鼠骨髓衍生巨噬细胞的制备
    1. 使用二氧化碳安乐死小鼠。在手术过程中,将剪刀和镊子放在70%乙醇中。在中央背部做一个小切口。从每个后腿的顶部剥下皮肤,然后从脚上往下。用剪刀切断髋骨后腿。仔细切断脚和髋骨,而不损害胫骨和股骨的完整性。通过在关节处切断分离胫骨和股骨。
    2. 从胫骨和股骨去除多余的肌肉。用镊子握住骨头并用70%乙醇喷雾。
    3. 将骨头放在含有P / S的冰冷DMEM的无菌管中。将管保持在冰上。
    4. 使用镊子,将骨头浸泡在70%乙醇中几秒钟。等待多余的乙醇滴下,然后用剪刀切割骨骼的各个末端以暴露骨髓腔。
    5. 使用连接到含有25ml冰冷的完全DMEM培养基(参见食谱)的50ml注射器上的25G针头冲洗骨髓,到无菌的50ml规格的离心管中。将样品通过40μm的细胞过滤器过滤到冰上新鲜的50ml管中。
      注意:在良好的细胞培养实践之后,所有液体步骤必须使用无菌和无热原材料在层流罩下进行,以避免巨噬细胞的活化。
    6. 在450℃离心细胞4分钟4分钟。
    7. 通过一次移动反转管,弃去上清液。将细胞重悬于含有50ng / ml M-CSF的冰冷的完全DMEM中
  2. 培养巨噬细胞
    1. 在含有M-CSF的完全DMEM中计数细胞并将细胞数调节至〜7×10 6 / ml。在最终体积为10ml的含有M-CSF的完全DMEM的培养皿中加入〜7×10 5个细胞。在37℃,5%CO 2培养箱中孵育培养皿。
    2. 在第3天,向每个板中加入5ml新鲜预热(37℃)的含有50ng / ml M-CSF的完全DMEM。

  3. 收获巨噬细胞
    1. 在第6天,丢弃培养上清液。立即加入10毫升冰冷的细胞培养级无菌1x PBS,不含钙和镁,含有5 mM EDTA的pH 7.4。孵化培养皿〜3分钟冰。
    2. 以30°C的角度握住培养皿。使用移液器控制器和无菌移液管冲洗培养皿及其内容物。将分离的细胞转移到含有转运体积的完全DMEM的六分之一的50ml无菌管中。
    3. 在4℃下以200×g离心管8分钟。
    4. 使用细胞计数室或自动细胞计数器计数细胞。将细胞浓度调整至1.25×10 6细胞/ ml。

  4. 电镀巨噬细胞
    使用多通道移液管或层流罩下的自动细胞分配器将100μl细胞移至96孔透明的底部黑色板中(图1)。在培养箱(37℃,5%CO 2)中孵育细胞过夜以确保细胞附着。
    注意:细胞应分配在孔的边缘以具有均匀分布。必须避免板块的振动或旋转,以防止孔中某些区域的细胞积聚(图2)。


    图1.液体的吸入和分布应在板的边缘进行。 将板与表面保持约30°的角度,并将液体吸入或分配到井中时,将多面吸管以120°的角度握住,以避免细胞脱落。


    图2.来自井的7×7瓦片图像的示例。 A和B.显示可能由细胞电镀,板振动/旋转和/或吸入/分散引起的异质细胞分布。 C.显示均匀的细胞分布。图像显示DAPI染色的巨噬细胞荧光核
  5. 治疗巨噬细胞
    1. 可以用优化的浓度的药物抑制剂预处理细胞或治疗优化的持续时间。在预处理的情况下,用预热的37℃完全DMEM洗涤孔,并加入100μl完整的DMEM。
      注意:
      1. 介质的抽吸和分散步骤必须在井的边缘进行,以避免分离单元(图2)。
      2. 按照该方案,进行选择性药理学抑制剂的双重连续稀释以确定初级巨噬细胞的无毒浓度。在实验的时间过程中使用适当的生物化学和/或细胞实验方法测定药物抑制剂在确定浓度下的效力。我们可以推荐使用1μM星形孢菌素(阴性对照)诱导巨噬细胞死亡和10-50ng / ml M-CSF(阳性对照)诱导巨噬细胞存活和增殖。
    2. 在100μl完整的DMEM中用感兴趣的刺激物处理细胞。孵育细胞在37℃,5%CO 2 注意:孵化的持续时间需要针对每个单独的实验装置进行优化。应该注意的是,在细胞培养基培养48小时后,巨噬细胞数量基本减少。

  6. 固定,染色和洗涤步骤
    1. 使用多通道移液管或自动洗板器吸出细胞上清液。
    2. 在1x PBS,pH 7.4中分配50μl4%PFA(参见食谱),并在室温下孵育平板10分钟。
    3. 使用多通道移液器或自动洗板机,用200μl1x PBS洗涤孔两次。
    4. 吸取上清液,分配50μl染色溶液。使用染色溶液(见食谱)在黑暗的地方孵育平板10分钟。
    5. 使用多通道移液器或自动洗板机,用200μl1x PBS冲洗两次。
    6. 密封板用粘合板手动密封或使用自动化板材封口机。

  7. 图像采集
    1. 用70%乙醇清洗每个96孔板的底部。
    2. 使用配备40x物镜的高含量显微镜,每孔获取49张图像,其中包括7 x 7图像,彼此间隔200μm。

  8. 用核和F-肌动蛋白染色计数细胞
    1. Metaexpress的细胞评分对象功能用于分割和定义所有细胞核(DAPI通道)。作为阳性标记,使用细胞质染色(鬼笔环肽通道)(图3A)。 “全核”和“正标记”参数的近似最小和最大宽度设置为5μm和30μm(图3B)。
      注意:可以使用ImageJ,Icy或Cell-profiler进行图像分析,这是免费的图像分析程序。
    2. 执行合并细胞核和细胞质对象的逻辑操作,以将细胞定义为算法中的对象(图3B)。
    3. 从图像中除去具有与图像相邻的细胞质的细胞,仅在视野中计数细胞(图3B)。
    4. 细胞计数作为图像分析管线的输出数据。


      图3.图像处理和对象分割管道。 :一种。未处理的DAPI,鬼笔环肽和复合图像; B.显示细胞核和细胞质的物体分割。细胞通过合并细胞核和细胞质二值图像来定义。在用于量化细胞数量的最终处理图像中移除接界细胞。

数据分析

每个独立的实验应该用三个技术或生物重复进行。为了汇集独立实验,如果使用药理学抑制剂,则可以将数据归一化为未处理状态下的细胞计数或载体处理条件下的细胞计数。条形图和适当的统计测试可用于表示数据。

食谱

  1. 完成DMEM细胞培养基
    50ml FBS
    5 ml HEPES
    5 ml P / S
    500毫升DMEM
    使用0.22μm过滤器和50 ml注射器将FBS,HEPES和P / S过滤到DMEM中
  2. 4%PFA溶液
    45毫升ddH 2 O O 1 N NaOH
    1 N HCl
    5 ml 10x PBS
    2克PFA
    热ddH 2 O至60℃。在化学罩下搅拌的同时,将2g PFA加入管中。加入几滴1N NaOH。等待解决方案变得清晰。加入10倍PBS,使用pH计和1N HCl调节pH至7.4。使用0.22μm和50 ml注射器过滤溶液。等分并储存于-20°C
  3. 5%皂苷溶液
    50ml 1x DPBS
    2.5克皂苷
    通过搅拌溶解皂苷。使用0.22μm和50 ml注射器过滤溶液。等分并储存于-20°C
  4. 染色溶液
    5μg/ ml DAPI(核酸染色)(库存浓度5mg / ml)
    1 U / ml鬼笔环肽(F-肌动蛋白染色)(原料浓度200 U / ml)
    0.05%(w / v)皂苷(原料浓度5%,w / v)
    1x DPBS,pH 7.4

致谢

感谢Dimitri Monreau和Cansel Ustunel Eren协助高分辨率显微镜和Slavica Masina对手稿的批判性阅读。我们感谢NCCR Geneva Access平台为高含量显微镜实验提供设备。这项工作由瑞士国家研究基金(FNRS 310030-153204和IZRJZ3_164176,N.F.),关节炎研究所(iAR)和COST行动(CM1307 SEFRI:C14.0070,N.F.)的资助提供资助。该方案根据Eren等人(2016)中描述的方案改编。

参考

  1. EDTA。  (2006)。 Cold Spring Harbor Protoc pdb.rec8030。
  2. Eren,RO,Reverte,M.,Rossi,M.,Hartley,MA,Castiglioni,P.,Prevel,F.,Martin,R.,Desponds,C.,Lye,LF,Drexler,SK,Reith, ,Beverley,SM,Ronet,C。和Fasel,N。(2016)。哺乳动物的先天性免疫应答利什曼原虫病毒RNA病毒增加巨噬细胞存活以促进寄生虫持久性。细胞宿主微生物 20(3) :318-328。
  3. Ginhoux,F。和Jung,S.(2014)。单核细胞和巨噬细胞:发育途径和组织体内平衡。 Nat Rev Immunol 14(6):392-404。
  4. Medzhitov,R。(2008)。起源和生理角色的炎症。自然 454(7203):428-435。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Eren, R. and Fasel, N. (2017). Macrophage Survival Assay Using High Content Microscopy. Bio-protocol 7(16): e2509. DOI: 10.21769/BioProtoc.2509.
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