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Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites
从卵巢癌腹水中分离单核细胞群   

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Abstract

Ovarian cancer is one of the most fatal tumors in women. Due to a lack of symptoms and adequate screening methods, patients are diagnosed at advanced stages with extensive tumor burden (Jelovac and Armstrong, 2011). Interestingly, ovarian cancer metastasis is generally found within the peritoneal cavity rather than other tissues (Lengyel, 2010; Tan et al., 2006). The reason behind this tissue tropism of the peritoneal cavity remains elusive. A prominent feature of this selectivity is ascites, the accumulation of fluid within the peritoneal cavity, containing, amongst others, immune cells, tumor cells and various soluble factors that can be involved in the progression of ovarian cancer (Kipps et al., 2013). The protocol described here is used to isolate mononuclear cells from ascites to study the functionality of the immune system within the peritoneal cavity.

Keywords: Ovarian cancer(卵巢癌), Ascites(腹水), Fluorescent activated cell sorting(荧光激活细胞分选), Mononuclear cells(单核细胞), Dendrititc cells(树突状细胞), Monocytes(单核细胞), Myeloid-derived suppressive cells(髓源性抑制细胞), BDCA1+CD14+ cells(BDCA1+CD14+细胞)

Background

Gradient centrifugation using LymphoprepTM is a standard protocol to isolate peripheral blood mononuclear cells (PBMCs). We slightly adjusted the protocol, regarding the sample preparation and amount of washing steps, in order to isolate mononuclear cells from ascites.

Materials and Reagents

  1. 50 ml tubes (Greiner Bio One International, catalog number: 227261 )
  2. Cell strainer 100 μm (Corning, Falcon®, catalog number: 352360 )
  3. 5 ml pipette (VWR, catalog number: VWR612-3702 )
  4. 10 ml pipette (VWR, catalog number: VWR612-3700 )
  5. 25 ml pipette (VWR, catalog number: VWR612-3697 )
  6. 225 ml tubes (Corning, Falcon®, catalog number: 352075 )
  7. 5 ml polypropylene round-bottom tube (Corning, Falcon®, catalog number: 352063 )
  8. Pre-separation filter 30 μm (Miltenyi Biotec, catalog number: 130-041-407 )
  9. Türk’s solution (EMD Millipore, catalog number: 1092770100 )
  10. LymphoprepTM (Alere Technologies, Axis-Shield, catalog number: 1114547 )
  11. Trypan blue stain (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  12. FcR blocking reagent (Miltenyi Biotec, catalog number: 120-000-442 )
  13. Antibodies
    1. Anti-CD45-V450 (BD, BD Biosciences, catalog number: 560367 )
    2. Anti-CD19-FITC (Dako Cytomation, catalog number: F0768 )
    3. Anti-CD20-FITC (BD, BD Biosciences, catalog number: 345792 )
    4. Anti-CD56-FITC (BD, BD Biosciences, catalog number: 345811 )
    5. Anti-CD1c-PE (BDCA1) (Miltenyi Biotec, catalog number: 130-090-508 )
    6. Anti-CD14-PerCP (BD, BD Biosciences, catalog number: 345786 )
    7. Anti-HLA-DR-PE-Cy7 (BD, BD Biosciences, catalog number: 335830 )
    8. Anti-CD4-APC-Cy7 (BD, BD Biosciences, catalog number: 557871 )
    9. Anti-CD3-BV510 (BD, BD Biosciences, catalog number: 563109 )
  14. Ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 1084211000 )
  15. Bovine serum albumin (BSA) (Roche Diagnostics, catalog number: 10735108001 )
  16. Phosphate-buffered saline (PBS) (Braun Melsungen, catalog number: 362 3140 )
  17. X-VIVO 15 (Lonza, catalog number: BE02-060Q )
  18. Antibiotic/Antimycotic (AA) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  19. Human serum (HS) (Bloodbank Rivierenland)
  20. 0.5 M EDTA (see Recipes)
  21. 10% BSA (see Recipes)
  22. Diluting buffer (see Recipes)
  23. Wash buffer (see Recipes)
  24. Staining buffer (see Recipes)
  25. Medium (see Recipes)

Equipment

  1. Centrifuge (Hettich Instruments, model: Rotanta 460R )
  2. Cell sorter (BD, model: FACS Aria II)
  3. Magnetic stirrer (Heidolph instruments, model: MR Hei-Mix S )

Procedure

  1. Isolation of immune cells from ascites
    1. Take four 50 ml tubes and put a 100 μm cell strainer on top of each of them.
    2. Pipette the ascites on top of the cell strainer and collect the ascites in the 50 ml tube underneath. Transfer the filtered ascites to a 225 ml Falcon tube.
      Note: Filtering the ascites removes tissue, fat or blood clumps that could interfere with cell isolation and cell sorting.
    3. Centrifuge at 4 °C:
      Maximum acceleration
      Running time 15 min at 490 x g
      Maximum break
    4. Pour away the supernatant. Be careful, as the pellet detaches easily when high amounts of red blood cells are present.
    5. Resuspend the pellet in diluting solution and count the cells with Türk’s solution (use Türks:cells in a ratio of 10:1). Dilute the cell suspension to a concentration of 100 x 106 cells per 10 ml in diluting buffer. Add 10 ml of the cell suspension per 50 ml tube and complete to 30 ml with diluting solution.
      Note: Do not put more than 100 x 106 ascites-derived cells on top of the LymphoprepTM layer, as this may interfere with the separation of the cells.
    6. Pipette 10 ml LymphoprepTM underneath the diluted cells (Figures 1A-1C). Take up 12 ml LymphoprepTM using a 10 ml pipette. Let the pipette tip slide carefully along the side of the tube until it reaches the bottom. Detach the pipette from the pipette boy and wait for the LymphoprepTM to leak out. Pull out the pipette until the tip reaches the top of the LymphoprepTM layer. Put your finger on top of the pipette to close the system and remove the pipette from the tube. There should be approximately 2 ml of LymphoprepTM left in the pipette.
    7. Centrifuge at room temperature:
      Acceleration time 240 sec (R/1)
      Running time 20 min at 970 x g
      No break
    8. Carefully remove the interphase with a 5 ml pipette and collect it in a new 50 ml tube (Figure 1D). Bring the volume to 50 ml with diluting solution.
      Note: Be careful when removing the interphase. Hold the pipette tip slightly above the interphase. This prevents removing the LymphoprepTM solution along with the interphase layer.
    9. Centrifuge at room temperature:
      Maximum acceleration
      Running time 10 min at 640 x g
      Maximum break
    10. Pour away the supernatant and resuspend the pellet in 50 ml washing solution.
    11. Centrifuge at 4 °C:
      Maximum acceleration
      Running time 5 min at 490 x g
      Maximum break
    12. Pool the cells into one 50 ml tube and repeat washing with washing solution until the supernatant is clear.
      Note: It usually takes 1 or 2 washing steps until the supernatant is clear.
    13. Count the cells with trypan blue stain (dilute cells and trypan blue in a ratio of 1:1).


      Figure 1. Density gradient centrifugation. A. Use a 10 ml pipette to take up 12 ml LymphoprepTM. Slide the pipette tip along the tube wall until it reaches the bottom. B. Disconnect the pipette from the pipette boy and wait for the LymphorpepTM to leak out. C. Carefully and slowly remove the pipette from the tube. D. After centrifugation, immune cells accumulate in different layers, based on their density. Mononuclear cells (B cells, T cells, NK cells, monocytes) are located in the interphase, between the diluting buffer and the LymphoprepTM. Polymorphonuclear cells (granulocytes) can be found on top of the red blood cells, which settle at the bottom of the tube.

  2. FACS staining
    1. Centrifuge cells at 4 °C:
      Maximum acceleration
      Running time 5 min at 490 x g
      Maximum break
    2. Pour away the supernatant and dilute cells with staining buffer to a concentration of 50 x 106 cells/ml.
    3. Transfer 50 x 106 cells to a new 50 ml tube.
    4. Centrifuge at 4 °C:
      Maximum acceleration
      Running time 5 min at 490 x g
      Maximum break
    5. Pour away the supernatant. The remaining liquid has a volume of approximately 300 μl.
    6. Add 50 μl FcR blocking reagent (1 μl per 1 x 106 cells) and incubate for 5 min at 4 °C.
    7. Add 1 ml staining buffer and centrifuge at 4 °C:
      Maximum acceleration
      Running time 5 min at 490 x g
      Maximum break
    8. Add 12.5 μl of anti-HLA-DR-PE-Cy7 (0.25 μl antibody per 1 x 106 cells) and 25 μl of each of the remaining antibodies antibodies (0.5 μl antibody per 1 x 106 cells): anti-CD45-V450, anti-CD19-FITC, anti-CD20-FITC, anti-CD56-FITC, anit-CD1c-PE, anti-CD14-PerCP, anti-HLA-DR-PE-Cy7, anti-CD4-APC-Cy7, anti-CD3-BV510.
    9. Incubate for 20 min at 4 °C and in the dark.
    10. Add 10 ml staining buffer and centrifuge at 4 °C:
      Maximum acceleration
      Running time 5 min at 490 x g
      Maximum break
    11. Pour away the supernatant and repeat step B10 two more times.
    12. Resuspend cells in 1 ml staining buffer.
      Note: For cell sorting, do not use higher concentrations than 50 x 106 cells/ml, as this will result in too many events per seconds that are measured by the FACS Aria II and interferes with proper sorting.
    13. Place a 30 μm filter on top of a new 5 ml polypropylene round-bottom tube and pass the cells trough the filter into the new tube.
      Note: Passing the cells through a 30 μm filter removes cell clumps that could obstruct the nozzle of the cell sorter.
    14. Prepare three 5 ml polypropylene round-bottom tubes with 1 ml medium to collect the cells in after sorting.

  3. Cell sorting
    1. Cell sorting is performed at room temperature. The collection tubes within the sorter are kept at 4 °C.
      Note: Keep the cells before and after sorting on ice to increase cell viability.
    2. Select the 85 μm nozzle on the FACS Aria II, to reduce the pressure on sorted cells.
    3. The following gating strategy was used to sort the different cell populations (Figure 2).
    4. Collect the cells in a 5 ml polypropylene round-bottom tube with 1 ml medium.
      Note: Shake the round-bottom tube before collecting the cells to cover the surface of the tube with medium. This prevents the cells from sticking to the tube wall.


      Figure 2. Gating strategy to isolate immune cell subsets using fluorescent activated cell sorting. A. Gating on immune cells on the FSC/SSC plot; B and C. Selection of single cells; D. Gating on immune cell population, which is CD45-V450 positive (G4), thereby excluding B cells and NK cells. E. Different immune cell populations can be identified, based on the expression of HLA-DR-PE-Cy7 and CD14-PerCP. Myeloid-derived suppressor cells are CD14+HLA-DR-. F. From gate G5, CD3+CD4+ T cells were identified and isolated. G. Gate G6 (HLA-DR+CD14-) was used to identify BDCA1+ cells. H. CD14+HLA-DR+ cells (P2) can be further subdivided into CD14+ and BDCA1+CD14+ cells.

Data analysis

The isolated immune cell subsets from ascites can be used for different experiments, like mixed lymphocyte reaction, suppressor assays or antigen uptake assays, as published in Bakdash et al., 2016.

Notes

  1. As large amounts of ascites can be collected, it is important to spin it down first in order to enrich the cells and dissolve them in a smaller volume.
  2. LymphoprepTM must be protected from light and stored at room temperature to ensure good separation.
  3. Keep diluting solution and EDTA at RT.
  4. 10% BSA and washing solution need to be stored at 4 °C.
  5. When preparing 0.5 M EDTA it is important to note that the EDTA only dissolves at a pH of 8.
  6. FITC is used as a dump channel. The three FITC labeled antibodies CD19, CD20 and CD56 are used to exclude B cells and NK cells.

Recipes

Note: All buffers and dilutions are stable when stored at the recommended temperature and used under sterile conditions.

  1. 0.5 M EDTA (store at RT)
    186.1 g EDTA
    800 ml dH2O
    Stir vigorously on a magnetic stirrer and adjust pH to 8.0
    Bring volume to 1 L with dH2O
  2. 10% BSA (store at 4 °C)
    10 g BSA
    100 ml PBS
  3. Diluting buffer (store at RT)
    5 ml 0.5 M EDTA
    500 ml PBS
  4. Wash buffer (store at 4 °C)
    5 ml 0.5 M EDTA
    5 ml 10% BSA
    500 ml PBS
  5. Staining buffer (store at 4 °C)
    PBS
    1% BSA
    1% EDTA
  6. Medium (store at 4 °C)
    100 ml X-VIVO 15
    1 ml AA
    2 ml HS

Acknowledgments

This work was supported by grant 951.03.002 from the Netherlands Organization for Scientific Research (NWO) and the Netherlands Institute of Regenerative Medicine (NIRM, FES0908). IJMdV is recipient of NWO Vici grant 918.14.655.
The procedure for the isolation of mononuclear cells from ascites was adapted from the standard protocol used for the isolation of PBMCs (Bakdash et al, 2016).

References

  1. Bakdash, G., Buschow, S. I., Gorris, M. A., Halilovic, A., Hato, S. V., Skold, A. E., Schreibelt, G., Sittig, S. P., Torensma, R., Duiveman-de Boer, T., Schroder, C., Smits, E. L, Figdor, C. G., de Vries, I. J. (2016). Expansion of a BDCA1+CD14+ myeloid cell population in melanoma patients may attenuate the efficacy of dendritic cell vaccines. Cancer Res 76(15): 4332-4346.
  2. Jelovac, D. and Armstrong, D. K. (2011). Recent progress in the diagnosis and treatment of ovarian cancer. CA Cancer J Clin 61(3): 183-203.
  3. Kipps, E., Tan, D. S. and Kaye, S. B. (2013). Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer 13(4): 273-282.
  4. Lengyel, E. (2010). Ovarian cancer development and metastasis. Am J Pathol 177(3): 1053-1064.
  5. Tan, D. S., Agarwal, R. and Kaye, S. B. (2006). Mechanisms of transcoelomic metastasis in ovarian cancer. Lancet Oncol 7(11): 925-934.

简介

卵巢癌是女性最致命的肿瘤之一。由于缺乏症状和适当的筛查方法,患者被诊断为具有广泛肿瘤负担的晚期阶段(Jelovac和Armstrong,2011)。有趣的是,卵巢癌转移通常发生在腹腔内,而不是其他组织(Lengyel,2010; Tan等人,2006)。腹膜腔组织向性背后的原因仍然难以捉摸。这种选择性的一个突出特征是腹水,腹膜内的液体积累,其中包含免疫细胞,肿瘤细胞和可参与卵巢癌进展的各种可溶性因子(Kipps等,。,2013)。这里描述的方案用于从腹水中分离单核细胞以研究腹膜腔内免疫系统的功能。

使用Lymphoprep 的梯度离心是分离外周血单核细胞(PBMC)的标准方案。我们稍微调整了方案,关于样品制备和洗涤步骤的数量,以便从腹水中分离单核细胞。

关键字:卵巢癌, 腹水, 荧光激活细胞分选, 单核细胞, 树突状细胞, 单核细胞, 髓源性抑制细胞, BDCA1+CD14+细胞

材料和试剂

  1. (Greiner Bio One International,目录号:227261)
  2. 细胞过滤器100μm(Corning,Falcon ®,目录号:352360)
  3. 5ml移液器(VWR,目录号:VWR612-3702)
  4. 10 ml移液器(VWR,目录号:VWR612-3700)
  5. 25毫升移液器(VWR,目录号:VWR612-3697)
  6. 225ml管(Corning,Falcon ®,目录号:352075)
  7. 5毫升聚丙烯圆底管(Corning,Falcon ®,目录号:352063)
  8. 预分离过滤器30μm(Miltenyi Biotec,目录号:130-041-407)
  9. Türk的解决方案(EMD Millipore,目录号:1092770100)
  10. Lymphoprep TM (Alere Technologies,Axis-Shield,目录号:1114547)
  11. 台盼蓝染色(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
  12. FcR阻断试剂(Miltenyi Biotec,目录号:120-000-442)
  13. 抗体
    1. 抗CD45-V450(BD,BD Biosciences,目录号:560367)
    2. 抗CD19-FITC(Dako Cytomation,目录号:F0768)
    3. 抗CD20-FITC(BD,BD Biosciences,目录号:345792)
    4. 抗CD56-FITC(BD,BD Biosciences,目录号:345811)
    5. 抗CD1c-PE(BDCA1)(Miltenyi Biotec,目录号:130-090-508)
    6. 抗CD14-PerCP(BD,BD Biosciences,目录号:345786)
    7. 抗HLA-DR-PE-Cy7(BD,BD Biosciences,目录号:335830)
    8. 抗CD4-APC-Cy7(BD,BD Biosciences,目录号:557871)
    9. 抗CD3-BV510(BD,BD Biosciences,目录号:563109)
  14. 乙二胺四乙酸(EDTA)(EMD Millipore,目录号:1084211000)
  15. 牛血清白蛋白(BSA)(Roche Diagnostics,目录号:10735108001)
  16. 磷酸盐缓冲盐水(PBS)(Braun Melsungen,目录号:362 3140)
  17. X-VIVO 15(Lonza,目录号:BE02-060Q)
  18. 抗生素/抗真菌药(AA)(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
  19. 人血清(HS)(Bloodbank Rivierenland)
  20. 0.5 M EDTA(参见食谱)
  21. 10%BSA(参见食谱)
  22. 稀释缓冲液(见配方)
  23. 洗涤缓冲液(见配方)
  24. 染色缓冲液(见配方)
  25. 中等(见配方)

设备

  1. 离心机(Hettich Instruments,型号:Rotanta 460R)
  2. 细胞分选机(BD,型号:FACS Aria II)
  3. 磁力搅拌器(Heidolph乐器,型号:MR Hei-Mix S)

程序

  1. 从腹水中分离免疫细胞
    1. 取四个50毫升管,并在其上面放置一个100微米的细胞过滤器
    2. 移出细胞过滤器顶部的腹水并收集下面的50ml管中的腹水。将过滤的腹水转移到225ml Falcon管中。
      注意:过滤腹水会去除可能干扰细胞分离和细胞分选的组织,脂肪或血块。
    3. 在4°C离心:
      最大加速度
      运行时间在490 x g 15分钟 最大断点
    4. 倒出上清液。注意,当存在大量红细胞时,颗粒容易脱离
    5. 将沉淀重悬在稀释溶液中,用Türk溶液计数细胞(使用Türks:细胞比例为10:1)。将稀释缓冲液中的每10ml稀释细胞悬浮液至浓度为100×10 6个细胞。每50ml管加入10ml细胞悬浮液,用稀释液完成30 ml。
      注意:不要在Lymphoprep TM 层的顶部放置超过100 x 10 6 的腹水衍生细胞,因为这可能会干扰细胞的分离
    6. 在稀释的细胞下移液10 ml Lymphoprep TM(图1A-1C)。使用10ml移液管吸取12 ml Lymphoprep TM 。让吸管尖端沿着管的侧面小心滑动,直到其到达底部。从移液器男孩中取出移液器,然后等待Lymphoprep TM 泄漏出来。拉出移液管直到尖端到达Lymphoprep TM 层的顶部。将手指放在移液器顶部以关闭系统,并从管中取出移液器。在移液器中应该有大约2ml的Lymphoprep TM
    7. 室温离心:
      加速时间240秒(R/1)
      运行时间20分钟在970
      不破坏
    8. 用5ml移液管小心地移除间期,并将其收集在新的50ml管中(图1D)。用稀释溶液将体积减至50ml 注意:删除界面时要小心。握住吸液管尖端略高于界面。这样可以防止与间期层一起去除Lymphoprep TM 溶液。
    9. 室温离心:
      最大加速度
      运行时间10分钟,640 x x
      最大断点
    10. 倒出上清液,将沉淀物重新悬浮于50ml洗涤液中
    11. 在4°C离心:
      最大加速度
      运行时间在490
      的5分钟 最大断点
    12. 将细胞收集到一个50ml的管中,并用洗涤液重复洗涤,直到上清澄清 注意:通常需要1或2个洗涤步骤,直到上清液清除。
    13. 用台盼蓝染色(稀释细胞和台盼蓝比例为1:1)计数细胞。


      图1.密度梯度离心。 A.使用10ml移液管吸收12 ml Lymphoprep TM。沿着管壁滑动吸头,直到达到底部。 B.从吸管男孩断开移液器,等待Lymphorpep TM 泄漏出来。 C.小心地慢慢取出移液管。 D.离心后,根据其密度,免疫细胞积累在不同的层中。单核细胞(B细胞,T细胞,NK细胞,单核细胞)位于稀释缓冲液和Lymphoprep TM之间的间期。多形核细胞(粒细胞)可以在红细胞的顶部找到,其位于管底部。

  2. FACS染色
    1. 4℃离心细胞:
      最大加速度
      运行时间在490
      的5分钟 最大断点
    2. 倒出上清液,用染色缓冲液稀释细胞至浓度为50×10 6细胞/ml。
    3. 将50 x 10 6细胞转移到新的50ml管中。
    4. 在4°C离心:
      最大加速度
      运行时间在490
      的5分钟 最大断点
    5. 倒出上清液。剩余液体的体积约为300μl
    6. 加入50μlFcR封闭试剂(每1×10 6个细胞1μl),并在4℃下孵育5分钟。
    7. 加入1 ml染色缓冲液,并在4°C离心:
      最大加速度
      运行时间在490
      的5分钟 最大断点
    8. 加入12.5μl抗HLA-DR-PE-Cy7(每1×10 6个细胞0.25μl抗体)和25μl剩余的每种抗体抗体(0.5μl抗体每1×10 6个细胞) sup-6细胞):抗CD45-V450,抗CD19-FITC,抗-CD20-FITC,抗CD56-FITC,anit-CD1c-PE,抗CD14-PerCP,抗HLA- DR-PE-Cy7,抗CD4-APC-Cy7,抗CD3-BV510。
    9. 在4℃和黑暗中孵育20分钟。
    10. 加入10 ml染色缓冲液,并在4°C离心:
      最大加速度
      运行时间在490
      的5分钟 最大断点
    11. 倒出上清液,重复步骤B10两次。
    12. 将细胞重悬于1ml染色缓冲液中 注意:对于细胞分选,不要使用比50×10 6细胞/ml更高的浓度,因为这将导致由FACS Aria II测量的每秒钟太多的事件和干扰正确排序。
    13. 在一个新的5毫升聚丙烯圆底管的顶部放置一个30微米的过滤器,并将细胞通过过滤器通入新管中。
      注意:将细胞通过30μm过滤器可以去除可能阻碍细胞分选机喷嘴的细胞团。
    14. 用1ml培养基准备三个5ml聚丙烯圆底管,分选后收集细胞
  3. 细胞分选
    1. 细胞分选在室温下进行。分拣机内的收集管保持在4°C 注意:在冰上分选之前和之后保留细胞以增加细胞活力。
    2. 选择FACS Aria II上的85μm喷嘴,以减少分选细胞的压力。
    3. 使用以下门控策略对不同的细胞群进行分类(图2)
    4. 用1 ml培养基将细胞收集在5ml聚丙烯圆底管中 注意:在收集细胞之前摇动圆底管,用介质覆盖管表面。这样可防止细胞粘附在管壁上。


      图2.使用荧光激活细胞分选分离免疫细胞亚群的门控策略。 A.在FSC/SSC图上的免疫细胞门控; B和C.单细胞选择; D.对免疫细胞群进行门控,其为CD45-V450阳性(G4),从而排除B细胞和NK细胞。可以根据HLA-DR-PE-Cy7和CD14-PerCP的表达鉴定不同的免疫细胞群体。骨髓来源的抑制细胞是CD14抗体,HLA-DR 。 F.从门G5,鉴定并分离CD3 + CD4 + T细胞。 G.GG G6(HLA-DR + CD14 - )用于鉴定BDCA1 + 细胞。细胞(P2)可以进一步细分为CD14 + 和BDCA1 sup> + CD14 + 单元格。

数据分析

来自腹水的分离的免疫细胞亚群可用于不同的实验,如混合淋巴细胞反应,抑制试验或抗原摄取测定法,如Bakdash等人,2016年所公布。

笔记

  1. 由于可以收集大量的腹水,因此首先将其旋转以便使细胞富集并将其溶解在较小体积中。
  2. Lymphoprep TM 必须防止光照,并在室温下储存,以确保良好的分离。
  3. 在室温下保持稀释溶液和EDTA
  4. 10%BSA和洗涤液需要在4℃下储存。
  5. 当制备0.5M EDTA时,重要的是要注意,EDTA仅溶于pH为8.
  6. FITC用作转储通道。三种FITC标记抗体CD19,CD20和CD56用于排除B细胞和NK细胞

食谱

注意:所有缓冲液和稀释液在推荐温度下储存时都是稳定的,并在无菌条件下使用。

  1. 0.5 M EDTA(储存于室温)
    186.1g EDTA
    800ml dH 2 O O
    在磁力搅拌器上剧烈搅拌并调节pH至8.0
    带dH <2> O
    将音量调至1L
  2. 10%BSA(4℃储存)
    10克BSA
    100毫升PBS
  3. 稀释缓冲液(在RT存储)
    5 ml 0.5 M EDTA
    500毫升PBS
  4. 洗涤缓冲液(在4℃下储存)
    5 ml 0.5 M EDTA
    5 ml 10%BSA
    500毫升PBS
  5. 染色缓冲液(储存于4°C)
    PBS
    1%BSA
    1%EDTA
  6. 中等(储存于4°C)
    100 ml X-VIVO 15
    1 ml AA
    2毫升HS

致谢

这项工作得到荷兰科学研究组织(NWO)和荷兰再生医学研究所(NIRM,FES0908)的授权951.03.002的支持。 IJMdV是NWO Vici授权的收件人918.14.655。
从用于分离PBMC的标准方案改变从腹水中分离单核细胞的程序(Bakdash等人,2016)。

参考文献

  1. Bakdash,G.,Buschow,SI,Gorris,MA,Halilovic,A.,Hato,SV,Skold,AE,Schreibelt,G.,Sittig,SP,Torensma,R.,Duiveman-de Boer,T.,Schroder, C.,Smits,E.L,Figdor,CG,de Vries,IJ(2016)。  扩增黑素瘤患者的BDCA1 + CD14 + 骨髓细胞群体可能会减弱树突状细胞疫苗的功效。 em> Cancer Res 76(15):4332-4346。
  2. Jelovac,D.和Armstrong,DK(2011)。卵巢癌的诊断和治疗方面的最新进展癌症J Clin 61(3):183-203。
  3. Kipps,E.,Tan,DS和Kaye,SB(2013)。  满足卵巢癌腹水的挑战:治疗和研究的新途径。 Nat Rev Cancer 13(4):273-282。
  4. Lengyel,E.(2010)。卵巢癌发展和转移。 Am J Pathol 177(3):1053-1064。
  5. Tan,DS,Agarwal,R.和Kaye,SB(2006)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/17081918"target = "_blank">卵巢癌转移瘤的机制。"柳叶刀Oncol"7(11):925-934。
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引用:Wefers, C., Bakdash, G., Moreno Martin, M., Duiveman-de Boer, T., Torensma, R., Massuger, L. F. and de Vries, I. M. (2017). Isolation of Mononuclear Cell Populations from Ovarian Carcinoma Ascites. Bio-protocol 7(7): e2219. DOI: 10.21769/BioProtoc.2219.
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