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Macrophage Phagocytosis Assay of Staphylococcus aureus by Flow Cytometry
通过流式细胞术进行葡萄球菌属巨噬细胞吞噬作用的测量   

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Abstract

This protocol describes a straightforward technique to evaluate the phagocytotic capacity of murine macrophages for Staphylococcus aureus (S. aureus). By staining S. aureus with Hexidium Iodide and staining murine bone marrow-derived macrophages (BMDMs) with FITC, the macrophage bacterial up-taking ability can be rapidly analyzed by flow cytometry. S. aureus is a Gram-positive bacteria causing severe human and animal infections. Host immune cells such as macrophages serve to eliminate S. aureus by phagocytosing the pathogen and save the host from life-threatening diseases. Study of host macrophage ability to phagocytose S. aureus is important for understanding the host-pathogen interaction and can help to elucidate the pathogenesis of S. aureus infection. This protocol may also be applied for macrophage phagocytotic assay of other gram-positive bacteria.

Materials and Reagents

  1. Cell line-L929 (ATCC, catalog number: CCL-1 )
  2. Staphylococcus aureus strain-Sanger 476 (Wellcome Trust Sanger Institute, catalog number: MSSA476 )
  3. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 11965-092 )
  4. L-Glutamine (Life Technologies, Gibco®, catalog number: 25030-081 )
  5. Penicillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-148 )
  6. Fetal bovine serum (Hyclone, catalog number: SH30070.03 )
  7. DPBS (Life Technologies, Gibco®, catalog number: 14190 )
  8. EDTA (Sigma-Aldrich, catalog number: E6758 )
  9. Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200-056 )
  10. ACK lysis buffer (Life Technologies, Gibco®, catalog number: A10492-01 )
  11. Tissue-culture water (Hyclone, catalog number: SH3052902 )
  12. Tryptic Soy Broth powder (TSB) (BD, catalog number: 211768 )
  13. Hexidium Iodide (Life Technologies, InvitrogenTM, catalog number: H7593 )
  14. FITC-anti-mouse-F4/80 (BioLegend, catalog number: 123107 )
  15. DMEM complete media with 10% FBS (D10) (see Recipes)
  16. Supernatant of L-cell cultural media (L-sup) (see Recipes)
  17. Bone marrow macrophage medium (BMM medium) (see Recipes)
  18. 500 mM-EDTA (see Recipes)
  19. 5 mM-EDTA (see Recipes)
  20. TSB (see Recipes)
  21. Hexidium Iodide stock (see Recipes)

Equipment

  1. 26G needle (Sigma-Aldrich, catalog number: Z192392-100 EA)
  2. Syringe (Terumo Medical Corporation, catalog number: SS-30L )
  3. Cell culture dish (Corning, catalog number: 430293 )
  4. 6-Well Plate (Falcon, catalog number: 351146 )
  5. 75 cm flask (Corning, catalog number: 430641 )
  6. Vacuum filtration system (Corning, catalog number: 430758 )
  7. 50 ml Corning tube (Corning, catalog number: 430290 )
  8. 5 ml flow cytometry tube (BD, catalog number: 352054 )
  9. Bacterial incubator shaker
  10. CO2 incubator
  11. Sorvall RT7 centrifuge
  12. Flow cytometer (FACSCanto)

Software

  1. FlowJo

Procedure

  1. Differentiation of bone-marrow derived macrophage (BMDM)
    1. Euthanize three mice by CO2. Sterilize the abdomen and hind legs of the mice with 70% ethanol.
    2. Make an incision in the midline of the abdomen, and expose the hind limbs by clipping outward. Remove all muscle from the bones and cut bones at both ends.
    3. Flush out bone marrow progenitor cells from bones using DPBS by syringe with a 26G needle. Flush 3-5 times for each bone by 2 ml DPBS. Spin down cells in Sorval RT7 centrifuge (4 °C, 1,500 rpm, 5 min).
    4. After aspirating out the supernatant using vacuum suction, treat cells by 5 ml ACK lysis buffer to lyse the erythrocytes (1 min, room temperature).
    5. Next, add 45 ml of DPBS to the lysed cells to balance the osmotic pressure.
    6. Spin cells down again, count cells by hemocytometer, and adjust them to a concentration of 5 x 105 cells/ml in BMM medium. Seed 10 ml cells in each 10-cm tissue culture dish. Expect to yield 1 x 108 of bone marrow cells from three mice.
    7. Culture the cells for 3 days in CO2 incubator at 37 °C. Then change for fresh BMM medium and culture for another 4 days (10 ml/dish).
    8. The macrophages are now ready for functional assay.
    9. Place the dishes in a sterile tissue culture hood, and wash the dishes twice with 5 ml cold DPBS.
    10. Add 2 ml 5 mM-EDTA (room temperature) to each dish and wait around 5 min to detach the cells. Add 5 ml D10 to each dish and harvest all the cells. Expect to yield ~5 x 106 of BMDMs per dish.
    11. Using this preparation, more than 95% of the differentiated cells should be macrophages based on F4/80 and CD11b expression. Analyze the expression of these markers by routine flow cytometry techniques. Choose different dyes for each marker (e.g. FITC for F4/80 and APC for CD11b).

  2. Culture of S. aureus to exponential period
    1. One day before the experiment, inoculate S. aureus into 3 ml TSB in a bio-safety cabinet and culture the bacteria overnight in a bacterial incubator shaker (37 °C, 225 rpm). S. aureus is a bio-safety level 2 pathogen which can cause severe human infections.
    2. On the next morning, dilute 100 μl overnight culture into 10 ml new TSB. The approximate OD600 is around 0.1. Shake the bacteria approximately 2 hours until OD600 reach around 0.8 (37 °C, 225 rpm). OD600 of 1.0 is approximately 1.5 x 109 CFU/ml.
    3. Spin down the bacteria (4 °C, 3,000 rpm, 10 min) and wash twice with 50 ml cold DPBS.
    4. Adjust bacterial concentration to 5 x 108/ml in DPBS. Place the bacteria on ice before assay.

  3. Stain S. aureus with Hexidium Iodide
    1. Aliquot 1 ml S. aureus (5 x 108/ml) into a 50 ml Corning tube.
    2. Add 20 μl Hexidium Iodide stock and mix it well.
    3. Incubate the bacteria for 15 min in darkness (room temperature).
    4. Wash twice with 50 ml cold DPBS (4 °C, 3,000 rpm, 10 min).
    5. Re-suspend the bacteria in BMM medium at the concentration of 2 x 107/ml. The bacteria are ready for assay. Use them right away.

  4. Macrophage phagocytosis assay
    One day before the assay, seed 2 x 106 BMDMs into single wells in a 6-well plate. Apply the multiplicity of infection (MOI) 10 for S. aureus infection.
    1. Briefly, on the day of assay remove the old medium and replace it with new BMM medium containing S. aureus (2 x 107/ml) to each well.
    2.  Quickly spin the 6-well plate (room temperature, 500 rpm, 5 min). Incubate the cells in a CO2 incubator for 30 min to allow bacterial uptake by BMDMs.
    3. Then add 200 μl cold Trypsin-EDTA to the cells and incubate for 10 min at room temperature without agitation to remove residual bacteria at macrophage surface.
      Note: Trypsin-EDTA solution treatment is to remove the excess bacteria. Prolonged treatment may also affect BMDM adherence, so the treating time shouldn’t exceed 10 min. Trypsin-EDTA treatment will not interfere with F4/80 analysis.
    4. Wash the plate three times with DPBS, using 2ml DPBS for each wash.
    5. Add 0.5 ml 5mM-EDTA (room temperature) to the well to detach the cells in about 5 min and then 2 ml D10. Harvest the BMDMs by spinning (4 °C, 1,500 rpm, 5 min). Wash cells in 50 ml DPBS and re-suspend cells in 10 ml D10. Count the cells by hemocytometer.

  5. Stain macrophages with FITC-anti-mouse-F4/80
    1. Add 1 x 106 BMDMs from procedure D to 5 ml flow cytometry tube and spin down in Sorval RT7 centrifuge (4 °C, 1,500 rpm, 5 min). Aspirate the supernatant. Re-suspend cells in 100 μl DPBS. Three additional control tubes are required for flow cytometry analysis, including one blank tube without fluorescence dye, one FITC-anti-mouse-F4/80 single staining tube and one Hexidium Iodide single staining tube.
    2. Add 1 μl FITC-anti-mouse-F4/80 to the tube following manufacturer’s instruction (final concentration: 5 μg/ml). And incubate 20 min in darkness without agitation (room temperature).
    3. Then add 500 μl DPBS. The cells are ready for flow cytometry assay.

  6. Flow cytometry
    Routine flow cytometry techniques are applied for phagocytosis analysis following manufacturer’s instruction. In a FACSCanto machine, the fluorescence produced from Hexidium Iodide staining falls into FL2 channel (Excitation 488/Emmision 575) and fluorescence produced from FITC staining falls into FL1 channel (Excitation 488/Emmision 519). Results can be analyzed by FlowJo. The performer should be familiar with FlowJo software to analyze 4-color flow cytometry data. Briefly, open the flow cytometry data in FlowJo software. First, plot FSC and SSC in X-axis and Y-axis. Then gate single live cells. Finally plot FITC and PE in X-axis and Y-axis of the single live cells.


    Figure 1. Practical example of a bone marrow-derived macrophage (BMDM) phagocytosis experiment. Blank: BMDMs without both FITC-anti-F4/80 staining and bacterial uptake. FITC: BMDMs with FITC-anti-F4/80 staining but without bacterial uptake. Hexidium Iodide: BMDMs with bacterial uptake but without FITC-anti-F4/80 staining. BMDM-phagocytosis: BMDMs with both FITC-anti-F4/80 staining and bacterial uptake.

Recipes

  1. DMEM complete media with 10% FBS (D10)
    440 ml DMEM
    50 ml FBS
    5 ml L-Glutamine (Stocking concentration: 200 mM)
    5 ml penicillin-streptomycin (stocking concentration: 10,000 U/ml)
    Stored at 4 °C
  2. Supernatant of L-cell cultural media (L-sup)
    Seed 5 x 105 L-929 cells in 75 cm flask in 55 ml D10
    Culture the cells for more than one week
    Culturing medium was harvested and filtered through vacuum filtration system and frozen down and stored at -20 °C, and use them within 6 months
  3. BMM medium
    350 ml D10
    150 ml L-sup
    Stored at 4 °C
  4. 500 mM-EDTA
    146 g EDTA dissolved in tissue-culture water, adjust pH to 8.0
  5. 5 mM-EDTA
    5 ml 500 mM-EDTA
    495 ml DPBS
    Stored at 4 °C
  6. TSB
    Dissolve 30 g Tryptic Soy Broth powder into deionized water, and autoclave at 121 °C for 30 min
    Stored at room temperature
  7. Hexidium Iodide stock (5 mg/ml)
    Dissolve 5mg Hexidium Iodide in 1 ml DMSO in darkness
    Aliquot and stored at -20 °C

Acknowledgments

This work was supported by NIH R01-AI068804. Dr. Fowler was supported by Mid Career Mentoring Award K24-AI093969. The fluorescence staining of Staphylococcus aureus was adapted from Mason et al. (1998), and differentiation of bone marrow-derived macrophages was adapted from Weischenfeldt and Porse (2008).

References

  1. Mason, D. J., Shanmuganathan, S., Mortimer, F. C. and Gant, V. A. (1998). A fluorescent Gram stain for flow cytometry and epifluorescence microscopy. Appl Environ Microbiol 64(7): 2681-2685.
  2. Weischenfeldt, J. and Porse, B. (2008). Bone marrow-derived macrophages (BMM): isolation and applications. CSH Protoc 2008: pdb prot5080.
  3. Yan, Q., Sharma-Kuinkel, B. K., Deshmukh, H., Tsalik, E. L., Cyr, D. D., Lucas, J., Woods, C. W., Scott, W. K., Sempowski, G. D., Thaden, J., Rude, T. H., Ahn, S. H. and Fowler, V. G., Jr. (2014). Dusp3 and Psme3 are associated with murine susceptibility to Staphylococcus aureus infection and human sepsis. PLoS Pathog 10(6): e1004149.

简介

该协议描述了评估小鼠巨噬细胞对金黄色葡萄球菌(金黄色葡萄球菌)的吞噬能力的直接技术。 通过染色。 金黄色葡萄球菌与碘化己锭染色并用FITC染色鼠骨髓衍生的巨噬细胞(BMDM),可以通过流式细胞术快速分析巨噬细胞细菌摄取能力。 aureus 是一种革兰氏阳性菌,引起严重的人类和动物感染。 宿主免疫细胞例如巨噬细胞用于消除S。 金黄色葡萄球菌通过吞噬病原体并将宿主从危及生命的疾病中拯救。 宿主巨噬细胞吞噬能力的研究。 金黄色葡萄球菌对于理解宿主 - 病原体相互作用是重要的,并且可以帮助阐明S的发病机制。 aureus 感染。 该方案也可应用于其他革兰氏阳性菌的巨噬细胞吞噬测定。

材料和试剂

  1. 细胞系-L929(ATCC,目录号:CCL-1)
  2. 金黄色葡萄球菌菌株Sanger 476(Wellcome Trust Sanger Institute,目录号:MSSA476)
  3. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:11965-092)
  4. L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-081)
  5. 青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140-148)
  6. 胎牛血清(Hyclone,目录号:SH30070.03)
  7. DPBS(Life Technologies,Gibco ,目录号:14190)
  8. EDTA(Sigma-Aldrich,目录号:E6758)
  9. 胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25200-056)
  10. ACK裂解缓冲液(Life Technologies,Gibco ,目录号:A10492-01)
  11. 组织培养水(Hyclone,目录号:SH3052902)
  12. 胰蛋白酶大豆肉汤粉(TSB)(BD,目录号:211768)
  13. 碘化己啶(Life Technologies,Invitrogen TM,目录号:H7593)
  14. FITC-抗小鼠F4/80(BioLegend,目录号:123107)
  15. 具有10%FBS(D10)的DMEM完全培养基(参见配方)
  16. L细胞培养基上清液(L-sup)(参见配方)
  17. 骨髓巨噬细胞培养基(BMM培养基)(参见配方)
  18. 500 mM EDTA(见配方)
  19. 5 mM EDTA(见配方)
  20. TSB(参见配方)
  21. 碘化己锭(见配方)

设备

  1. 26G针(Sigma-Aldrich,目录号:Z192392-100EA)
  2. 注射器(Terumo Medical Corporation,目录号:SS-30L)
  3. 细胞培养皿(Corning,目录号:430293)
  4. 6孔板(Falcon,目录号:351146)
  5. 75cm烧瓶(Corning,目录号:430641)
  6. 真空过滤系统(Corning,目录号:430758)
  7. 50ml康宁管(Corning,目录号:430290)
  8. 5ml流式细胞术管(BD,目录号:352054)
  9. 细菌孵箱摇床
  10. CO <2>孵化器
  11. Sorvall RT7离心机
  12. 流式细胞仪(FACSCanto)

软件

  1. FlowJo

程序

  1. 骨髓来源的巨噬细胞(BMDM)的分化
    1. 通过CO 2安乐死三只小鼠。 用70%乙醇灭菌小鼠的腹部和后腿。
    2. 在腹部的中线做一个切口,暴露后脑 肢体。 删除所有的肌肉从骨头和切 骨头在两端。
    3. 排出骨髓祖细胞 骨头使用DPBS通过注射器用26G针。 每次冲洗3-5次 骨。 在Sorval RT7离心机中旋转细胞(4℃,1,500   rpm,5分钟)。
    4. 使用真空吸出上清液后 抽吸,通过5ml ACK裂解缓冲液处理细胞以裂解红细胞 (1分钟,室温)。
    5. 接下来,向裂解的细胞中加入45ml DPBS以平衡渗透压。
    6. 再次旋转细胞,通过血细胞计数器计数细胞,并调整它们   在BMM培养基中浓度为5×10 5个细胞/ml。 种子10ml细胞 在每个10-cm组织培养皿中。 预期产生1×10 8个骨 来自三只小鼠的骨髓细胞
    7. 在CO 2培养箱中在37℃下培养细胞3天。 然后换新鲜的BMM培养基并培养 另外4天(10ml /皿)。
    8. 巨噬细胞现在准备用于功能测定。
    9. 将盘放入无菌组织培养罩,用5ml冷DPBS洗涤两次。
    10. 向每个皿中加入2ml 5mM-EDTA(室温),并等待约5   min以分离细胞。 添加5毫升D10每个菜,收获所有的 细胞。 预期每个培养皿产生〜5 x 10 6 个BMDM
    11. 使用这个 准备,超过95%的分化细胞应该 基于F4/80和CD11b表达的巨噬细胞。 分析表达式 的这些标记物通过常规流式细胞术技术。 选择不同 染料(例如对于F4/80的FITC和对于CD11b的APC)。

  2. 文化。 aureus 指数期间
    1. 实验前一天,接种S. aureus 转化为3ml TSB 生物安全柜和细菌在细菌中过夜培养 培养摇床(37℃,225rpm)。


      aureus 是一种生物安全水平2 病原体,其可以引起严重的人类感染。
    2. 接下来 早晨,稀释100微升过夜培养到10毫升新的TSB。 的 近似OD 600大约为0.1。 摇动细菌约2 直到OD 600达到约0.8(37℃,225rpm)。 OD 600的1.0 约1.5×10 9 CFU/ml
    3. 旋转细菌(4℃,3,000rpm,10分钟),并用50ml冷DPBS洗涤两次
    4. 在DPBS中将细菌浓度调节至5×10 8 s/ml。 测定前将细菌放在冰上。

  3. Stain 。 aureus 与Hexidium Iodide
    1. 等分1ml em。 金黄色葡萄球菌(5×10 8 /ml)加入到50ml康宁管中。
    2. 加入20μl碘化铱股票并混合好。
    3. 在黑暗中(室温)孵育细菌15分钟。
    4. 用50ml冷DPBS洗涤两次(4℃,3,000rpm,10分钟)。
    5. 重悬细菌在BMM培养基中浓度为2× 10 /ml。 细菌准备用于测定。 立即使用它们。

  4. 巨噬细胞吞噬作用测定
    在测定前一天,将2×10 6个BMDM接种到6孔板中的单个孔中。 对于S应用感染复数(MOI)10。 aureus 感染。
    1. 简而言之,在测定当天,取出旧培养基并更换 新的含有S的BMM培养基。 aureus (2×10 7/u/ml)
    2.  快速旋转6孔板(室温,500rpm,5分钟)。 孵育细胞在CO 2培养箱中30分钟,以允许细菌 被BMDMs吸收。
    3. 然后加入200μl冷胰蛋白酶-EDTA的细胞 并在室温下孵育10分钟,无需搅拌除去 巨噬细胞表面的残留细菌。
      注意:胰蛋白酶-EDTA 溶液处理是除去过量的细菌。 长期治疗   也可能影响BMDM的粘附性,因此处理时间不应超过 10分钟。 胰蛋白酶-EDTA处理不会干扰F4/80分析。
    4. 用DPBS洗涤板三次,每次洗涤使用2ml DPBS。
    5. 加入0.5ml 5mM-EDTA(室温)至孔中以分离 细胞在约5分钟,然后2ml D10。 通过纺纱收获BMDMs(4   1500rpm,5分钟)。 洗涤细胞在50 ml DPBS和重悬细胞   10ml D10。 通过血细胞计数器计数细胞。

  5. 用FITC-抗小鼠F4/80染色巨噬细胞
    1. 将来自程序D的1×10 6个BMDM加入到5ml流式细胞仪管中并旋转 在Sorval RT7离心机中(4℃,1,500rpm,5分钟)。 吸气 上清液。 重悬细胞在100微升DPBS。 三个附加控制 包括一个空白管,用于流式细胞术分析 无荧光染料,一次FITC-抗小鼠F4/80单染色 管和一个Hexidium Iodide单染色管
    2. 加入1μl FITC-抗小鼠F4/80加入到试管中 (终浓度:5μg/ml)。 并在黑暗中孵化20分钟 搅拌(室温)。
    3. 然后加入500μlDPBS。 细胞准备用于流式细胞术测定。

  6. 流式细胞术
    常规流式细胞术技术用于根据制造商的说明进行的吞噬分析。 在FACSCanto机器中,从碘化己啶染色产生的荧光落入FL2通道(激发488/Emmision 575),从FITC染色产生的荧光落入FL1通道(激发488/Emmision 519)。 结果可以由FlowJo分析。 表演者应该熟悉FlowJo软件来分析4色流 细胞计数数据。简而言之,在FlowJo软件中打开流式细胞术数据。首先,在X轴和Y轴上绘制FSC和SSC。然后门单个活细胞。最后在单个活细胞的X轴和Y轴上绘制FITC和PE

    图1.骨髓来源的巨噬细胞(BMDM)吞噬实验的实例。空白:没有FITC-抗F4/80染色和细菌摄取的BMDM。 FITC:具有FITC-抗F4/80染色但无细菌摄取的BMDM。碘化己啶:具有细菌摄取但没有FITC-抗F4/80染色的BMDM。 BMDM-吞噬作用:具有FITC-抗F4/80染色和细菌摄取的BMDM。

食谱

  1. 含有10%FBS(D10)的DMEM完全培养基 440 ml DMEM
    50ml FBS
    5ml L-谷氨酰胺(储存浓度:200mM) 5ml青霉素 - 链霉素(储存浓度:10,000U/ml) 储存在4°C
  2. L细胞培养基上清液(L-sup)
    种子在75ml烧瓶中的5×10 5 L-929细胞在55ml D10中 将细胞培养一周以上
    收获培养基并通过真空过滤系统过滤,冷冻并储存在-20℃下,并在6个月内使用它们。
  3. BMM介质
    350 ml D10
    150 ml L-sup
    储存在4°C
  4. 500 mM -EDTA
    将146g EDTA溶于组织培养水中,将pH调节至8.0
  5. 5mM-EDTA 5ml 500mM-EDTA
    495ml DPBS
    储存在4°C
  6. TSB
    将30g胰蛋白酶大豆肉汤粉末溶解在去离子水中,在121℃高压灭菌30分钟
    在室温下贮存
  7. 碘化己锭(5mg/ml)
    将5mg碘化己啶溶解在1ml DMSO中,溶于黑暗中
    等分并储存在-20°C

致谢

这项工作由NIH R01-AI068804支持。 Fowler博士受到中等职业指导奖K24-AI093969的支持。 金黄色葡萄球菌的荧光染色从Mason等人(1998)改编,并且 骨髓来源的巨噬细胞的分化改编自Weischenfeldt和Porse(2008)。

参考文献

  1. Mason,D.J.,Shanmuganathan,S.,Mortimer,F.C.and Gant,V.A。(1998)。 用于流式细胞术和落射荧光显微镜的荧光革兰氏染色。 Appl Environ Microbiol 64(7):2681-2685。
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  3. Yan,Q.,Sharma-Kuinkel,BK,Deshmukh,H.,Tsalik,EL,Cyr,DD,Lucas,J.,Woods,CW,Scott,WK,Sempowski,GD,Thaden, Ahn,SH和Fowler,VG,Jr。(2014)。 Dusp3和Psme3与对金黄色葡萄球菌感染和人类的小鼠易感性有关败血症。 PLoS Pathog 10(6):e1004149。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yan, Q., Ahn, S. H. and Fowler, V. G. (2015). Macrophage Phagocytosis Assay of Staphylococcus aureus by Flow Cytometry. Bio-protocol 5(4): e1406. DOI: 10.21769/BioProtoc.1406.
  2. Yan, Q., Sharma-Kuinkel, B. K., Deshmukh, H., Tsalik, E. L., Cyr, D. D., Lucas, J., Woods, C. W., Scott, W. K., Sempowski, G. D., Thaden, J., Rude, T. H., Ahn, S. H. and Fowler, V. G., Jr. (2014). Dusp3 and Psme3 are associated with murine susceptibility to Staphylococcus aureus infection and human sepsis. PLoS Pathog 10(6): e1004149.
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