参见作者原研究论文

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Dec 2007
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Isolation and Stimulation of Peritoneal Macrophages with Apoptotic Jurkat Cells to Produce IL-10
提取和利用凋亡Jurkat细胞诱导腹腔巨噬细胞分泌白介素-10   

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Abstract

Clearance of apoptotic cells by macrophages is critical to ensuring cellular homeostasis and suppression of autoimmunity. Macrophage recognition of apoptotic cells triggers an anti-inflammatory response, which is mediated by the release of IL-10, TGF-β etc. with concurrent inhibition of pro-inflammatory cytokines (such as TNFα, IL-12, IL-1β). To characterize cytokine profile produced by macrophages during phagocytosis of apoptotic cells, we developed an effective, more physiologic system using isolated murine peritoneal macrophages and T-lymphocyte cell line Jurkat as a source of apoptotic cells. Apoptosis of Jurkat cells is induced with staurosporine, a protein kinase C (PKC) inhibitor and detected by Annexin V/propidium iodide staining. This in vitro assay demonstrates that murine peritoneal macrophages produce large amounts of IL-10 following exposure to apoptotic Jurkat cells.

Keywords: Apoptosis (细胞凋亡), Macrophage (巨噬细胞), Jurkat (Jurkat), IL-10 (白介素-10), Phagocytosis (吞噬作用)

Background

Production of IL-10, a major immunoregulatory cytokine, by phagocytes during clearance of apoptotic cells is critical to ensuring cellular homeostasis and suppression of autoimmunity (Chung et al., 2007). Little is known about the regulatory mechanisms in this fundamental process. To elucidate the molecular mechanisms involved in the regulation of IL-10 gene expression in macrophages upon interaction with apoptotic cells, we developed this protocol to explore key regulators of IL-10 production induced by apoptotic cells.

Materials and Reagents

  1. 24-well plate (Corning, catalog number: 3563847)
  2. T-75 flask (Corning, catalog number: 431464)
  3. Falcon® 50 ml High Clarity PP Centrifuge Tube (Corning, catalog number: 352070)
  4. Posi-Click 1.7 ml microcentrifuge tube (Denville, catalog number: C2170)
  5. 10-ml syringe (BD, catalog number: 309695)
  6. 25 G and 18 G needles (BD)
  7. C57BL/6 mice (6-8 weeks old)
  8. Cell line: Jurkat, Clone E6-1 (ATCC, catalog number: TIB-152)
  9. Fetal Bovine Serum (Thermo Fisher, catalog number: 16000)
  10. RPMI 1640 Medium, GlutaMAXTM Supplement, and HEPES (Thermo Fisher, catalog number: 72400)
  11. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher, catalog number: 15140)
  12. GlutaMAXTM Supplement (Thermo Fisher, catalog number: 35050)
  13. Staurosporine (Sigma-Aldrich, catalog number: 19-123)
  14. DPBS, no calcium, no magnesium (Thermo Fisher, catalog number: 14190)
  15. FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, catalog number: 556547)
  16. Mouse IL-10 ELISA Set (BD, catalog number: 565252)
  17. BD BBL Dehydrated Culture Media (Fisher Scientific, catalog number: 211716)
  18. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D2560)
  19. 70% Ethanol
  20. Complete media for Jurkat T cells (see Recipe 1)
  21. 3% (w/v) Brewer thioglycollate medium (see Recipe 2)
  22. 1x Binding buffer(see Recipe 3)
  23. RPMI-1640-10 (see Recipe 4)
  24. 0.5 mg/ml Staurosporine (1,000x stock) (see Recipe 5)

Equipment

  1. Tissue culture hood (biosafety cabinet, Forma Scientific, model: 1284)
  2. 37 °C, 5% CO2 humidified incubator (Thermo Forma, model: 3110 Series II)
  3. Centrifuge (Thermo Scientific, ST8 Benchtop Centrifuge)
  4. Optical microscopy (Olympus, CK30)
  5. Flow cytometer (BD, FACSCalibur)
  6. Sterile Surgical Scissors (Miltex, MH5-302)
  7. Hemacytometer (Hausser Scientific, catalog number: 02-671-5)
  8. Autoclave

Software

  1. FlowJo software (7.6.1)
  2. GraphPad Prism

Procedure

  1. Subculturing Jurkat cell Line
    1. Transfer growing cells from T-75 flask to a 50 ml centrifuge tube followed by centrifugation at 300 x g for 5 min at room temperature.
    2. Remove and discard the supernatant (old growth medium from above the cell pellet) and resuspend the cell pellet with fresh complete growth medium.
    3. Add a proper volume of Jurkat cell suspension to a new T-75 flask containing 15 ml of complete growth medium with approximately 105 viable cells/flask and subculture every 3-4 days.
    4. Place the culture flask in a 37 °C, 5% CO2 humidified incubator.

  2. Inducing apoptosis of Jurkat cells
    1. Harvest exponentially growing Jurkat cells (initial concentration of 2 x 105 cells/ml, and final concentration of 6-8 x 105 cells /ml, usually two days after seeding) by centrifugation at 300 x g for 5 min (same procedure for Jurkat cells subculturing/harvesting/washing).
    2. Resuspend cells in fresh medium to a final concentration of 1 x 106 cells/ml and seed into a new T-75 flask. Another flask will be used as the negative control for non-induced cells (note that there would be two groups, one for apoptosis, and the other for non-apoptosis control group).
    3. Add staurosporine into Jurkat cell culture with a final concentration of 0.5 μg/ml, and incubate in a 37 °C, 5% CO2 humidified incubator for 6-8 h. Add the same volume of DMSO into control Jurkat T cell culture.
    4. Harvest cells for staining at different times (i.e., 3, 6, 12 h) after the addition of staurosporin/DMSO by transferring 2 ml of cell culture medium into a fresh Falcon tube and centrifugation. Keep culturing the rest of cells in T-75 flask in a 37 °C, 5% CO2 humidified incubator.

  3. Detection apoptosis of Jurkat cells with FITC Annexin V Apoptosis Detection Kit
    1.  Wash harvested staurosporine-treated Jurkat cells twice with cold DPBS to stop the reaction.
    2. Resuspend cells in 1x Binding buffer at a concentration of 5 x 106 cells/ml.
    3. Transfer 100 μl of the solution (~5 x 105 cells) to a new 1.7 ml microcentrifuge tube.
    4. Add 2 μl of FITC Annexin V and 2 μl of PI, gently vortex the cells and incubate for 15 min at room temperature or 30 min on ice, protecting from light under either condition.
    5. Add 400 μl of 1x Binding buffer to each tube, wash twice (centrifugation at 400 x g for 5 min) and analyze by flow cytometer within 1 h (Figure 1).


      Figure 1. Apoptosis of Jurkat cells induced by staurosporine. After 6 h-induction, the percentage of early- and late-apoptotic cells was quantified by flow cytometry analysis with Annexin V and propidium iodide (PI) staining. The numbers indicate the percentage of subpopulations.

  4. Intraperitoneal injection of thioglycollate
    1. Fill a 10-ml syringe with 3% Brewer thioglycollate medium with a 25 G needle.
    2. Inject 2 ml of solution per mouse into the peritoneal cavity, allowing inflammatory response to proceed for 4 days.

  5. Harvest and culture thioglycollate-elicited peritoneal macrophages
    1. Euthanize mouse with CO2 and clean abdomen with 70% ethanol.
    2. Make a small incision along the midline with sterile scissors and retract the abdominal skin manually to expose the transparent peritoneal wall.
    3. Fill a 10-ml syringe with 9 ml of DPBS or RPMI-1640-10 (Recipe 4), then inject harvest medium with a 25-G needle into the peritoneal cavity.
    4. Massage abdomen for ~30 s and recover peritoneal fluid without puncturing any abdominal organs using a 10 ml syringe with 18 G needle (~8 ml fluid could be recovered from per mouse) (Video 1).

      Video 1. Harvest peritoneal macrophages (Steps E2-E4)

    5. Remove the needle from the syringe and dispense peritoneal fluid into a 15 ml Centrifuge tube on ice (Cells should be kept cool throughout the procedure).
    6. Centrifuge the peritoneal cells in a refrigerated centrifuge for 5 min at 400 x g. Discard supernatant and resuspend the cell pellet in 5 ml of RPMI-1640-10 medium by gently pipetting up and down.
    7. Count cells using hemacytometer (with 1:10 dilution by mixing 100 μl of cell suspension with 900 μl of trypan blue).
    8. Adjust cell concentration with RPMI-1640-10 and seed into 24-well plate at 5 x 105 cells/well (500 μl of medium per well).
    9. Cells are allowed to adhere to the plate by culturing for 2-3 h in 37 °C, 5% CO2 humidified incubator. Non-adherent cells are removed by gently washing three times with 1 ml of warmed PBS (Remaining cells should be greater than 90% macrophages).

  6. Stimulate peritoneal macrophages with apoptotic Jurkat cells
    1. After overnight culture, media for peritoneal macrophages are changed with 500 μl of RPMI-1640 without FBS.
    2. Add 100 μl of induced and evaluated apoptotic Jurkat cells (suspended in RPMI-6140 without FBS, with a concentration of 25 x 106 cells/ml) into macrophage cultures at a 5:1 ratio.
    3. Harvest cell culture supernatants at 8 h, 16 h after apoptotic cell challenge.
    4. Centrifuge harvested samples in a refrigerated centrifuge for 10 min at 500 x g, and then carefully transfer cleared supernatant (~600 μl) to a new tube without disturbing cell pellet.

  7. Detection of IL-10 production by ELISA (Figure 2)


    Figure 2. Macrophages produce IL-10 in response to apoptotic cells. 5 x 105 thioglycollate-elicited peritoneal macrophages were stimulated with apoptotic Jurkat cells (AC) (5:1 ratio of AC to macrophages) for indicated times and analyzed for IL-10 production.

Data analysis

  1. Analysis was performed using FlowJo software (7.6.1). Average three independent replicates to obtain a single read to each sample.
  2. To evaluate apoptosis of Jurkat cells, use unstained negative control to determine the absolute boundaries for both Annexin V-FITC and propidium iodide (PI) positive cells. Be sure to compare data generated with control cells (DMSO treated cells) to that of the unstained negative control.
  3. For ELISA, subtract the value of blank sample to avoid background reads.
  4. Mean values and standard deviation (S.D.) were used as descriptive statistics. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test (P < 0.05 was considered statistically significant).

Recipes

  1. Complete media for Jurkat cells
    RPMI 1640 Medium, GlutaMAXTM Supplement, and HEPES (450 ml)
    50 ml (10%) Fetal Bovine Serum
    5 ml (100x) Penicillin-Streptomycin
    5 ml (100x) GlutaMAXTM Supplement
  2. 3% (w/v) Brewer thioglycollate medium
    1. Suspend 30 g of BD BBL Dehydrated Culture Media (Fisher scientific) in 1,000 ml of distilled water and autoclave to sterilize
    2. After cooling, aliquot to 50 ml Centrifuge Tubes, and could be stored in the dark at room temperature for 3 months
  3. 1x Binding buffer
    10x Annexin V Binding Buffer (component no. 51-66121E): 0.1 M HEPES/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2
    For a 1x working solution, dilute 1 part of the 10x Annexin V Binding Buffer to 9 parts of distilled water
    Note: 1x Binding buffer should be freshly prepared.
  4. RPMI-1640-10
    To prepare RPMI-1640-10, additionally supplement RPMI-1640 medium with 10% (v/v) fetal bovine serum
  5. 0.5 mg/ml Staurosporine (1,000x stock)
    1. Resolve 200 μg of Staurosporine (Sigma-Aldrich) in 400 μl Dimethyl sulfoxide (Sigma-Aldrich)
    2. Aliquot and store at -20 °C and thaw an aliquot before each use

Acknowledgments

This work was supported by an NIH grant (1R21AI110815) to X.M. This protocol was adapted from Chung EY et al. (2007).

Competing interests

The authors declare no conflicts of interest.

References

  1. Chung, E. Y., Liu, J., Homma, Y., Zhang, Y., Brendolan, A., Saggese, M., Han, J., Silverstein, R., Selleri, L. and Ma, X. (2007). Interleukin-10 expression in macrophages during phagocytosis of apoptotic cells is mediated by homeodomain proteins Pbx1 and Prep-1. Immunity 27(6): 952-964.

简介

巨噬细胞清除凋亡细胞对于确保细胞稳态和抑制自身免疫至关重要。巨噬细胞对凋亡细胞的识别触发了抗炎反应,该反应是由IL-10,TGF-β等的释放介导的。同时抑制促炎性细胞因子(例如TNFα,IL-12,IL-1β)。为了表征在凋亡细胞吞噬过程中巨噬细胞产生的细胞因子谱,我们开发了一种有效的,更生理的系统,使用了分离的鼠腹膜巨噬细胞和T淋巴细胞细胞系Jurkat作为凋亡细胞的来源。星形孢菌素是一种蛋白激酶C(PKC)抑制剂,可诱导Jurkat细胞凋亡,并通过Annexin V /碘化丙啶染色进行检测。此体外分析表明,鼠腹膜巨噬细胞暴露于凋亡的Jurkat细胞后会产生大量IL-10。

【背景】吞噬细胞在凋亡细胞清除过程中产生主要的免疫调节细胞因子IL-10,对确保细胞稳态和抑制自身免疫至关重要(Chung et al。,2007)。关于这一基本过程中的监管机制知之甚少。为了阐明与凋亡细胞相互作用后巨噬细胞中IL-10基因表达调控的分子机制,我们开发了该方案以探索凋亡细胞诱导的IL-10产生的关键调控因子。

关键字:细胞凋亡, 巨噬细胞, Jurkat, 白介素-10, 吞噬作用

材料和试剂

  1. 24孔板(Corning,目录号:3563847)
  2. T-75烧瓶(Corning,目录号:431464)
  3. Falcon ® 50 ml高清晰度PP离心管(Corning,目录号:352070)
  4. Posi-Click 1.7 ml微量离心管(Denville,目录号:C2170)
  5. 10毫升注射器(BD,货号:309695)
  6. 25 G和18 G针(BD)
  7. C57BL / 6小鼠(6-8周大)
  8. 细胞系:Jurkat,克隆E6-1(ATCC,目录号:TIB-152)
  9. 胎牛血清(Thermo Fisher,目录号:16000)
  10. RPMI 1640 Medium,GlutaMAX TM 补充剂和HEPES(Thermo Fisher,目录号:72400)
  11. 青霉素-链霉素(10,000 U / ml)(Thermo Fisher,目录号:15140)
  12. GlutaMAX TM 补充(Thermo Fisher,目录号:35050)
  13. 星形孢菌素(Sigma-Aldrich,目录号:19-123)
  14. DPBS,无钙,无镁(Thermo Fisher,目录号:14190)
  15. FITC膜联蛋白V细胞凋亡检测试剂盒I(BD Pharmingen,目录号:556547)
  16. 小鼠IL-10 ELISA试剂盒(BD,目录号:565252)
  17. BD BBL脱水培养基(Fisher Scientific,目录号:211716)
  18. 二甲基亚砜(Sigma-Aldrich,目录号:D2560)
  19. 70%乙醇
  20. Jurkat T细胞的完整培养基(请参见配方1)
  21. 3%(w / v)Brewer巯基乙酸盐培养基(请参见配方2)
  22. 1x绑定缓冲区(请参阅配方3)
  23. RPMI-1640-10(请参阅第4章)
  24. 0.5 mg / ml星形孢菌素(1,000x存量)(请参见第5条)

设备

  1. 组织培养罩(生物安全柜,Forma Scientific,型号:1284)
  2. 37°C,5%CO 2 加湿培养箱(Thermo Forma,型号:3110 Series II)
  3. 离心机(Thermo Scientific,ST8台式离心机)
  4. 光学显微镜(奥林巴斯,CK30)
  5. 流式细胞仪(BD,FACSCalibur)
  6. 无菌手术剪刀(Miltex,MH5-302)
  7. 血细胞计数器(Hausser Scientific,目录号:02-671-5)
  8. 高压灭菌器

软件

  1. FlowJo软件(7.6.1)
  2. GraphPad Prism

程序

  1. 传代Jurkat细胞系
    1. 将生长中的细胞从T-75烧瓶转移到50 ml离心管中,然后在室温下以300 x g 离心5分钟。
    2. 除去并丢弃上清液(细胞沉淀上方的旧生长培养基),然后用新鲜的完整生长培养基重悬细胞沉淀。
    3. 将适量的Jurkat细胞悬液添加到一个新的T-75烧瓶中,该烧瓶中装有15 ml完全生长培养基,每10天约有10 5 个活细胞/瓶,并每3-4天传代培养一次。
    4. 将培养瓶置于37°C,5%CO 2 湿润培养箱中。

  2. 诱导Jurkat细胞凋亡
    1. 通常在播种后两天收获指数生长的Jurkat细胞(初始浓度为2 x 10 5 细胞/ ml,最终浓度为6-8 x 10 5 细胞/ ml ),以300 xem 的速度离心5分钟(Jurkat细胞的继代培养/收获/洗涤步骤相同)。
    2. 将细胞重悬于新鲜培养基中至终浓度为1 x 10 6 细胞/ ml,并接种到新的T-75烧瓶中。另一个烧瓶将用作未诱导细胞的阴性对照(请注意,将有两组,一组用于凋亡,另一组用于非凋亡对照组)。
    3. 将星形孢菌素添加到Jurkat细胞培养物中,终浓度为0.5μg/ ml,并在37°C,5%CO 2 湿润培养箱中孵育6-8小时。将相同体积的DMSO加入对照Jurkat T细胞培养物中。
    4. 加入星形孢菌素/ DMSO后,通过将2 ml细胞培养基转移到新鲜的Falcon管中并离心,收获细胞以在不同时间(即,3、6、12 h)染色。继续在37°C,5%CO 2 湿润培养箱中于T-75烧瓶中培养其余细胞。

  3. FITC Annexin V凋亡检测试剂盒检测Jurkat细胞凋亡
    1. 用冷DPBS洗涤两次收获经星形孢菌素处理的Jurkat细胞,以终止反应。
    2. 在1x结合缓冲液中以5 x 10 6 细胞/ ml的浓度重悬细胞。
    3. 将100μl溶液(〜5 x 10 5 细胞)转移至新的1.7 ml微量离心管中
    4. 加入2μlFITC Annexin V和2μlPI,轻轻涡旋细胞,在室温下孵育15分钟或在冰上孵育30分钟,在任何一种情况下均避光。
    5. 向每个试管中加入400μl1x结合缓冲液,洗涤两次(以400 x g 离心5分钟),并在1小时内通过流式细胞仪进行分析(图1)。


      图1.星形孢菌素诱导的Jurkat细胞凋亡。诱导后6 h,通过Annexin V和碘化丙锭(PI)染色的流式细胞仪分析了早期和晚期凋亡细胞的百分比。 。数字表示亚种群的百分比。

  4. 腹腔注射巯基乙酸盐
    1. 用25 G针将10%的注射器充满3%Brewer巯基乙酸盐培养基。
    2. 每只小鼠将2 ml溶液注入腹膜腔,使炎症反应持续4天。

  5. 收获和培养巯基乙酸盐引起的腹膜巨噬细胞
    1. 用CO 2 对小鼠实施安乐死,并用70%的乙醇清洁腹部。
    2. 用无菌剪刀沿中线做一个小切口,然后手动缩回腹部皮肤,露出透明的腹膜壁。
    3. 用9 ml DPBS或RPMI-1640-10(配方4)填充10 ml注射器,然后用25 G针将收获培养基注入腹膜腔。
    4. 按摩腹部约30 s,并使用10毫升18 G针注射器恢复腹腔积液而不会刺破任何腹部器官(每只小鼠可回收约8毫升液)(视频1)。


      视频1.收获腹膜巨噬细胞(步骤E2-E4)

    5. 从注射器上取下针头,将腹膜液分配到冰上的15 ml离心管中(在整个过程中应保持细胞冷却)
    6. 在冷藏离心机中以400 x g 的速度离心腹膜细胞5分钟。弃去上清液,然后轻轻地上下移液,将细胞沉淀重悬于5 ml RPMI-1640-10培养基中。
    7. 使用血细胞计数器对细胞进行计数(将100μl细胞悬液与900μl锥虫蓝混合,以1:10稀释)。
    8. 用RPMI-1640-10调节细胞浓度,并以5 x 10 5 细胞/孔(每孔500μl培养基)的浓度接种到24孔板中。
    9. 通过在37°C,5%CO 2 湿润培养箱中培养2-3小时,使细胞粘附到板上。通过用1 ml加热的PBS轻轻洗涤3次来去除非贴壁细胞(剩余细胞应大于90%巨噬细胞)。

  6. 用凋亡的Jurkat细胞刺激腹膜巨噬细胞
    1. 过夜培养后,用500μl不含FBS的RPMI-1640更换腹膜巨噬细胞的培养基。
    2. 以5:1的比例将100μl诱导和评估的凋亡Jurkat细胞(悬浮在无FBS的RPMI-6140中,浓度为25 x 10 6 细胞/ ml)。
    3. 凋亡细胞攻击后8小时,16小时收获细胞培养上清液。
    4. 在冷藏离心机中以500 x g 离心收集的样品10分钟,然后小心地将澄清的上清液(〜600μl)转移到新试管中,而不会干扰细胞沉淀。

  7. 通过ELISA检测IL-10的产生(图2)


    图2.巨噬细胞响应凋亡细胞而产生IL-10。用凋亡的Jurkat细胞(AC)刺激5 x 10 5 巯基乙酸盐诱导的腹膜巨噬细胞(5:1)交流电与巨噬细胞的比例),并分析IL-10的产生。

数据分析

  1. 使用FlowJo软件(7.6.1)进行分析。平均三个独立的重复样本,以获得每个样品的单次读数。
  2. 要评估Jurkat细胞的凋亡,请使用未染色的阴性对照来确定膜联蛋白V-FITC和碘化丙啶(PI)阳性细胞的绝对边界。确保将对照细胞(DMSO处理过的细胞)产生的数据与未染色的阴性对照进行比较。
  3. 对于ELISA,请减去空白样品的值,以避免背景读数。
  4. 平均值和标准差(SD)用作描述性统计数据。两组之间的比较使用未配对的双尾学生 t 检验( P <0.05被认为具有统计学意义)。

菜谱

  1. Jurkat细胞的完整培养基
    RPMI 1640培养基,GlutaMAX TM 补充剂和HEPES(450毫升)
    50毫升(10%)胎牛血清
    5毫升(100x)青霉素-链霉素
    5毫升(100x)GlutaMAX TM 补品
  2. 3%(w / v)布鲁尔巯基乙酸盐培养基
    1. 将30 g BD BBL脱水培养基(Fisher science)悬浮在1000 ml蒸馏水中,并高压灭菌。
    2. 冷却后,等分至50 ml离心管中,可在室温下于黑暗中保存3个月
  3. 1个绑定缓冲区
    10x Annexin V结合缓冲液(组分编号51-66121E):0.1 M HEPES / NaOH(pH 7.4),1.4 M NaCl,25 mM CaCl 2
    对于1倍的工作溶液,请将1倍的10倍的Annexin V结合缓冲液稀释至9份的蒸馏水
    注意:应重新准备1x绑定缓冲区。
  4. RPMI-1640-10
    要准备RPMI-1640-10,请另外添加含10%(v / v)胎牛血清的RPMI-1640培养基
  5. 0.5 mg / ml星形孢菌素(1,000x存量)
    1. 在400μl二甲基亚砜(Sigma-Aldrich)中溶解200μg星形孢菌素(Sigma-Aldrich)
    2. 分装并保存在-20°C并在每次使用前解冻等分

致谢

这项工作得到了XM的NIH资助(1R21AI110815)的支持。该协议改编自Chung EY 等人。(2007年)。

利益争夺

作者宣称没有利益冲突。

参考文献

  1. 钟永安,刘健,霍马,Y。,张永健,布伦多兰,A。萨格斯,M。,韩健,西尔弗斯坦,R。,塞勒利,L和马X.(2007 )。凋亡细胞吞噬过程中巨噬细胞中白介素10的表达是由同源域蛋白Pbx1和Prep-1介导的。 免疫力 27(6):952-964。
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引用:Song, M. and Ma, X. (2019). Isolation and Stimulation of Peritoneal Macrophages with Apoptotic Jurkat Cells to Produce IL-10. Bio-protocol 9(24): e3467. DOI: 10.21769/BioProtoc.3467.
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