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In vitro Culture of Human PBMCs
人外周血单核细胞(PBMC)的体外培养   

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Abstract

Peripheral blood mononuclear cells (PBMCs) consist of chiefly of lymphocytes and monocytes. Purified PBMCs are used in vitro to evaluate a variety of functions of lymphocytes viz; a) proliferation to mitogenic (PHA, Con-A) stimulation, b) monitoring of prior sensitisation in antigen recall assays by scoring lymphocyte proliferation, c) immunophenotyping for surface markers as well as intracellular molecules in monocytes and lymphocytes etc. Activation of monocytes/macrophages by small molecules, cytokines and pathogen components can also be monitored. PBMCs can also be used for a variety of structural and functional studies for addressing issues in human immunology such as scoring for apoptosis and production of cytokines as well as other mediators in vitro.

Materials and Reagents

  1. Freshly collected heparinised blood
  2. Ficoll Histopaque (Sigma- Aldrich, catalog number: 10771 )
  3. Sterile PBS or Dulbecco's modified eagle medium (DMEM) (catalog number: P-04-03590 )
  4. Pencillin-streptomycin solution (Sigma- Aldrich, catalog number: P4333 )
  5. W. B. C. diluting fluid (Qualigen, catalog number: 42425 )
  6. Fetal bovine serum (Pan Biotech, catalog number: 1302-P100402 )
  7. Trypan blue (Mediatech, catalog number: 193 )
  8. DMEM supplemented with 1% of Pencillin-streptomycin solution and 10% FBS (see Recipes)

Equipment

  1. Centrifuge machine with swing-out bucket rotors (Eppendorf, catalog number: 5810 R )
  2. Heparin vials (BD Biosciences, catalog number: 367886 )
  3. Sterile 15 ml centrifuge tube
  4. Auto pipettes
  5. 200 µl and 1 ml tips
  6. 24 well cell culture plate (TPP Techno Plastic Products, catalog number: 20090123 )
  7. Haemocytometer
  8. Tissue culture hood
  9. CO2 incubator
  10. Microscope

Procedure

  1. Collect about 4 ml of human venous blood sample in heparinised vials and mix well by gently inverting the tubes several times.
  2. Isolate human PBMCs by gradient centrifugation using Ficoll-Histophaque.
  3. Wash cells (centrifuge at 100 x g for 10 min) with 10 ml of sterile DMEM (without FBS) twice.
    Note: Cold DMEM is not used routinely for washing lymphocytes from culture cavities while setting up cultures. Rather when monocytes bound tightly to plastic cavities are needed to be harvested pre-chilled DMEM can be used.
  4. Discard medium and re-suspend the cell pellet in 1 ml of sterile Dulbecco's modified eagle medium.
  5. Count cells by haemocytometer using W.B.C. diluting fluid: Add 10 µl of cell suspension to 190 µl of W.B.C. diluting fluid and mix well. Load the cell suspension in a haemocytometer and count the cells. Adjust cell concentration at 1 x 106 cells/ml with Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS. The approximate yield of cells from 4 ml of blood varies between 107-108.
  6. Seed 500 µl of cell suspension in a 24 well culture plate.
    Note: Monocytes in PBMCs get attached to the plastic in about 2-3 h when incubated at 37 °C. Longer incubation will result in firm attachement. Lymphocytes are not glass adherent and they will be mostly in suspension and can be removed by mildly flushing the wells with medium and/or buffer. Such treatment will keep the monocytes firmly attached to the surface of culture plates.
  7. Cells can be treated with different antigens for different period of times and the supernatants can be analysed for cytokine levels. The cells can be analysed for phenotypic change, apoptosis or proliferation.
    Note: PBMCs are primary cells and cannot be cultured for more than one passage under normal conditions. Lymphocytes of PBMCs can be made to proliferate in vitro by mitogens e.g., Phytohaemagglutin or Concanavin-A etc over a period to 72-96 h. Monocytes generally are end cells and do not proliferate. In absence of mitogens the proliferation of PBMCs will be negligible.

Recipes

  1. Dulbecco's modified eagle medium supplemented with 1% of Pencillin-streptomycin solution and 10% FBS

Technical notes

  1. Viability of the isolated PBMCs needs to be monitored add 200 µl of cell suspension to 200 µl of 0.4% of trypan blue solution and incubate for 15 min. Cheque for viability of cells by trypan blue staining and score under a microscope using haemocytometer (cells taking up a blue stain are dead cells).
  2. Calculate percentage viability as follows:
    Cell Viability = total Viable cells/total cells x 100
  3. Cell suspension having more than 95% viability should be used for culture.

Acknowledgments

The laboratory protocol was evolved over time in the senior authors’ laboratory using a template that was published in 1986 in Handbook of Experimental Immunology / edited by D. M. Weir; co-editors, L. A. Herzenberg, Caroline Blackwell, Leonore A. Herzenberg. Blackwell Scientific Publications. Institute of Life Sciences is funded by Department of Biotechnology, Govt of India and SP was supported by a fellowship grant from Indian Council of Medical Research.

References

  1. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V. and Ravindran, B. (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.

简介

外周血单核细胞(PBMC)主要由淋巴细胞和单核细胞组成。 纯化的PBMC在体外用于评价淋巴细胞的多种功能, a)增殖至促有丝分裂(PHA,Con-A)刺激,b)通过评估淋巴细胞增殖来监测抗原召回试验中的先前致敏,c)表面标志物以及单核细胞和淋巴细胞等中的细胞内分子的免疫表型。 也可以监测小分子,细胞因子和病原体组分的巨噬细胞。 PBMC还可以用于多种结构和功能研究,用于解决人免疫学中的问题,例如细胞凋亡评分和细胞因子以及其他介体的体外生产。

材料和试剂

  1. 新鲜收集的肝素化血液
  2. Ficoll Histopaque(Sigma-Aldrich,目录号:10771)
  3. 无菌PBS或Dulbecco改良的Eagle培养基(DMEM)(目录号:P-04-03590)
  4. 青霉素 - 链霉素溶液(Sigma-Aldrich,目录号:P4333)
  5. (Qualigen,目录号:42425)
  6. 胎牛血清(Pan Biotech,目录号:1302-P100402)
  7. 台盼蓝(Mediatech,目录号:193)
  8. 补充有1%青霉素 - 链霉素溶液和10%FBS的DMEM(参见Recipes)

设备

  1. 带有旋出式铲斗转子(Eppendorf,目录号:5810 R)的离心机
  2. 肝素小瓶(BD Biosciences,目录号:367886)
  3. 无菌15ml离心管
  4. 自动移液器
  5. 200μl和1 ml提示
  6. 24孔细胞培养板(TPP Techno Plastic Products,目录号:20090123)
  7. 血细胞计数器
  8. 组织培养罩
  9. CO <2>孵化器
  10. 显微镜

程序

  1. 在肝素化的小瓶中收集约4ml的人静脉血样品,并通过轻轻颠倒管子几次混合均匀。
  2. 通过使用Ficoll-Histophaque的梯度离心分离人PBMC
  3. 用10ml无菌DMEM(不含FBS)洗涤细胞(以100×g离心10分钟)两次。
    注意:在建立培养物时,冷DMEM通常不用于从培养腔清洗淋巴细胞。相反,当需要收获单核细胞紧密地结合到塑料空腔时,可以使用预冷的DMEM。
  4. 弃去培养基并将细胞沉淀重悬在1ml无菌Dulbecco's改良的Eagle培养基中。
  5. 通过血细胞计数器使用W.B.C.计数细胞稀释液:将10μl细胞悬浮液加入到190μl的W.B.C.稀释液和混合好。加载细胞悬浮液在血细胞计数器和计数细胞。用补充有1%青霉素 - 链霉素溶液和10%FBS的Dulbecco改良的Eagle培养基调整细胞浓度为1×10 6个细胞/ml。来自4ml血液的细胞的近似产率在10 -10 8 之间变化。
  6. 在24孔培养板中接种500μl细胞悬浮液 注意:当在37℃温育时,PBMC中的单核细胞在约2-3小时内附着到塑料上。更长的孵化将导致坚实的附件。淋巴细胞不是玻璃粘附的,并且它们将主要在悬浮液中,并且可以通过用培养基和/或缓冲液温和冲洗孔来除去。这种处理将使单核细胞牢固地附着于培养板的表面。
  7. 细胞可以用不同的抗原处理不同的时间段,并且可以分析上清液的细胞因子水平。可以分析细胞的表型变化,凋亡或增殖 注意:PBMC是原代细胞,并且在正常条件下不能培养超过一次传代。可以使PBMC的淋巴细胞在体外通过有丝分裂原例如,植物血细胞凝集素或Concanavin-A 等一段时间到72-96小时。 单核细胞通常是端细胞并且不增殖。 在没有促细胞分裂剂的情况下,PBMC的增殖将是可忽略的

食谱

  1. Dulbecco's改良的Eagle培养基,补充有1%的青霉素 - 链霉素溶液和10%FBS

技术说明

  1. 需要监测分离的PBMC的活力将200μl细胞悬浮液加入到200μl0.4%的台盼蓝溶液中并孵育15分钟。 通过台盼蓝染色检查细胞的活力,并在显微镜下使用血球计(细胞吸收蓝色染色的细胞是死细胞)得分。
  2. 计算百分比生存力如下:
    细胞活力=总活力细胞/总细胞×100
  3. 应该使用具有超过95%活力的细胞悬浮液进行培养。

致谢

实验室方案在高级作者实验室中使用1986年在Handbook of Experimental Immunology/edited by D.M.Weir中发表的模板随时间演变; 共同编辑,L. A. Herzenberg,Caroline Blackwell,Leonore A. Herzenberg。 Blackwell科学出版社。 生命科学研究所由生物技术部资助,印度政府,SP得到印度医学研究委员会的奖学金资助。

参考文献

  1. Panda,S.K.,Kumar,S.,Tupperwar,N.C.,Vaidya,T.,George,A.,Rath,S.,Bal,V.and Ravindran,B。(2012)。 Chitohexaose通过TLR4的交替途径激活巨噬细胞,阻断内毒素血症。 8(5):e1002717。
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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: © 2013 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. Panda, S. K. and Ravindran, B. (2013). In vitro Culture of Human PBMCs. Bio-protocol 3(3): e322. DOI: 10.21769/BioProtoc.322.
  2. Panda, S. K., Kumar, S., Tupperwar, N. C., Vaidya, T., George, A., Rath, S., Bal, V. and Ravindran, B. (2012). Chitohexaose activates macrophages by alternate pathway through TLR4 and blocks endotoxemia. PLoS Pathog 8(5): e1002717.
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Ana Ayala
Chungbuk National University
Hello, I isolated and cultured canine PBMC at 10^5 cells/well (in 100 μl) in 96 well plate and added Con A at 10 μg/ml for 48 hours. I did the reading with WST prolif. assay, but the reading show results of less formazan salt formation in my "stimulated" PBMC, compared with my control PBMC! Can it be that the concentration of Con A is too high for the cells and actually be toxic for them?
9/27/2017 12:32:34 AM Reply
Balachandran Ravindran
Infectious Disease Biology, Institute of Life Sciences, India

Hi The most likely possibility is toxicity of ConA at a concentration of 10ug/ml that you use. Some makes of ConA could be toxic at this concentration. The other possibility is viability of your cells. At the end of 48 hrs you should get >90% viable cells. Hope this helps

9/27/2017 5:09:41 AM


Ana Ayala
Chungbuk National University

I see. I will try proliferation with lower concentrations of Con A. Still the reading would be an accurate reading if I just use WST-1? or is it definitely just accurate with BrdU ELISA or Flow cytometry? Could it be that if I use WST-1 i need to incubate for longer time to have a proliferation reading, since it measures metabolism instead of the DNA synthesis measured through BrdU? As I am very new in the research field, I am truly thankful for your quick reply.

9/27/2017 5:45:02 PM


Snehal Raut
Graduate Student
Could you please tell me how to positively select PBMC (with FACS or any immunofluroscence markers)? I have understood after reading the protocols and research articles, lymphocytes are non-adherent. So from suspended culture, how to positively select let's say either CD4 or CD8 cells? After selection are these cells adherent or non-adherent?
9/12/2017 10:29:04 AM Reply
Ioanna Eleftheriadou
Aristotle University of Thessaloniki
How can I check PBMCs viability after treatment with a substance( carvacrol) ? I have tried MTT assay but I think that is not enough! So, i want to try with Trypan blue 0,4%. I have my cells in 96 well plate. After 24h incubation with carvacrol how can I check viability with trypan blue?
4/22/2016 7:17:25 AM Reply
Santosh Panda
Infectious Disease Biology, Institute of Life Sciences, India

Hi Loanna
You can remove some cells from the plate and can use trypans blue. otherwise you can also stain them by DAPI and check by FACS.
thanks

4/25/2016 7:02:51 AM


Ioanna Eleftheriadou
Aristotle University of Thessaloniki

Thank you very much. I have one more question: Do the PBMCs proliferate without mitogen?I seed 150000cells/well and incubate with a plant extract. 20 hours later, i count more cells. Which is the proliferation rate of PBMCs?

4/25/2016 2:54:02 PM


Santosh Panda
Infectious Disease Biology, Institute of Life Sciences, India

Hi
Loanna
My boss Dr. Ravindran answer this to a previous query. here it is, Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.
hope this will be helpful.
thanks

4/26/2016 6:18:48 AM


Shadia Nada
University of Toledo
is it possible to freeze the PBMC and what is the best media to be use for freezing
3/24/2015 9:23:43 AM Reply
Santosh Panda
Infectious Disease Biology, Institute of Life Sciences, India

Dear Shadia, Can you please clarify why you want to freeze??

3/26/2015 12:19:40 PM


Shadia Nada
University of Toledo

I’m purifying the PBMC from human blood and I use the cells right away I’d like to ask if I can freeze the rest of the cells for next experiment, and if I froze them, are they going to be function as the fresh cells if I thaw and use them again and what is the best media for freezing. Thank you for your help and time

3/26/2015 12:31:16 PM


Santosh Panda
Infectious Disease Biology, Institute of Life Sciences, India

We never tried in this way. If I have to advice you based on my experience it is not going to function physiologically equivalent to fresh cells. You can freeze them for DNA isolation or cell lysate preparation but not for any immune response study.

3/26/2015 12:35:27 PM


Thao Tran
Texas A&M
Mohamed,

Did you find way to make the cells to grow?

Thanks
2/11/2015 2:48:45 PM Reply
Balachandran Ravindran
Infectious Disease Biology, Institute of Life Sciences, India

Cells in PBMC constituting Monocytes and Lymphocytes are terminally differentiated cells normally. Monocytes do not divide and can only be differentiated to macrophages by adding cytokines into culture. Lymphocytes also will not divide unless activated by a variety of means. Under normal culture conditions cells will die over time.

2/11/2015 8:32:58 PM


Thao Tran
Texas A&M

I activated PBMC with either PHA 5 ug/ml or LPS 1 ug/ml. I used medium IMDM with 20% bovine serum. After 4 days, nothing worked, the cell population decreased! Now I increase the PHA and LPS up to 10 times concentration. I hope it will woek (after the first day, I checked but it still has not been working well). How long is the wait? Any one could provide me suggestions?

2/11/2015 8:49:07 PM


Balachandran Ravindran
Infectious Disease Biology, Institute of Life Sciences, India

Can you clarify when you say 'nothing worked?!' I guess there was no proliferation of cells PHA or LPS. How did you score proliferation? How sure you are about the quality of PHA or LPS? Adding 10 times more PHA or LPS could actually be toxic to cells.

2/19/2015 9:31:04 PM


Thao Tran
Texas A&M

I counted by hemocytometer. Over the time, the cell population decreased. I am using LPS from sigma and PHA ftom Gibco.

2/20/2015 6:03:30 AM


mohamed ashour
saddat
How can I set up culture from PBMC?

1- I separate blood using ficoll
2- used RPMI+ 10%FBS+ Lglu+ strep/pencellin
3- culture the cell in 96 well plate (10000 cells/well) and in flask at 1000000 cell/ml

the cells is not growing and the counting is down every day. How can I set up a culturing condition which could give me replicating PBMC to test these cells for drug developments


Regards,
7/24/2014 3:29:14 PM Reply