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Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been shown to be appropriately capable of differential activation in multiple studies.

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Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol
采用简单的逆流层析法纯化人单核细胞和淋巴细胞群

免疫学 > 免疫细胞分离 > 淋巴细胞
作者: Elizabeth V. Clarke
Elizabeth V. ClarkeAffiliation: Molecular Biology and Biochemistry Department, University of California, Irvine, CA, USA
Bio-protocol author page: a787
Marie E. Benoit
Marie E. BenoitAffiliation: Molecular Biology and Biochemistry Department, University of California, Irvine, CA, USA
Bio-protocol author page: a786
 and Andrea J. Tenner
Andrea J. TennerAffiliation: Molecular Biology and Biochemistry Department, University of California, Irvine, CA, USA
For correspondence: atenner@uci.edu
Bio-protocol author page: a788
Vol 3, Iss 23, 12/5/2013, 3929 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.981

[Abstract] Investigations of the activation processes involved in human monocytes and monocyte-derived macrophages and dendritic cells often required large numbers of cells that have not been possibly altered or activated by adherence to surfaces, by binding of antibodies to surface antigens during positive selection, or by release of activators by platelets or other non myeloid cells during isolation or co-culture. Human peripheral blood monocytes as well as lymphocytes from the same blood donor can be isolated by counterflow elutriation using a modification of the technique of Lionetti et al. (1980) as described previously (Bobak et al., 1986). From a unit of blood drawn into anticoagulant, 60-120 million monocytes can be obtained. These cells are not activated and have been shown to be appropriately capable of differential activation in multiple studies.
Keywords: Monocyte(单核细胞), Human(人类), Isolation(隔离), Elutriation(淘洗), Lymphocyte(淋巴细胞)

[Abstract]

Materials and Reagents

  1. Peripheral blood from normal human donor (450 ml)
  2. CPD AS-1 blood collection bags (Citrate-Phosphate-Dextrose, Adenine saline-1) (Baxter)
  3. 1x PBS-2mM EDTA
  4. Sterile milliQ H2O
  5. Lymphocyte separating medium (LSM) (MP Biomedicals, catalog number: 0 850494 )
  6. HBSS (Fisher Scientific, catalog number: 21023CV )
  7. Ethanol (70%, 1 liter)
  8. 10% Bleach (1 liter)
  9. 10% defined FBS (Hyclone, catalog number: SH30070.03 )
  10. 1% P/S (Mediatech, CellGro®, catalog number: 30-002-CIRF )
  11. 1% L-Gln (Mediatech, CellGro®, catalog number: 25-005-CI )
  12. Recombinant human IL-2 (Pepro Tech, catalog number: 200-02 )
  13. Recombinant human M-CSF (Pepro Tech, catalog number: 300-25 )
  14. 25% Albumin (Human), USP (Talecris Plasma Resources, catalog number: 13533-684-20 )
  15. Complete media (see Recipes)
  16. 1x sterile PBS (dilute 1:5 from 5x PBS pH 7.0 – 7.2) (see Recipes)
  17. Ammonium Chloride Potassium (ACK) buffer (see Recipes)
  18. 1x sterile Elutriation Buffer (see Recipes)
  19. 5x Phosphate Buffered Saline (see Recipes)

Equipment

  1. Extruder
  2. T75 flasks
  3. Nunc blue cap 50-ml polypropylene tubes (important as monocytes stick to other tubes lowing yields)
  4. Tubing clamps
  5. Scissors
  6. P1000 Pipetteman
  7. Petri dishes
  8. 250 ml plasma tube
  9. Isopropanol wipes, individually packaged
  10. Beckman JE6B Elutriation System and Rotor (Beckman Coulter, model: JE6B-IM-3) (November 1992)**
  11. Centrifuge (Sorvall, model: RT6000 or RC-3B )
  12. Stericup (Fisher Scientific, catalog number: SCGVU11RE )
    ** Beckman Coulter has newer models Avanti J-301 or J-26S XP with Elutriation Systems, but we have no experience with those.

Procedure

  1. Receive 450 ml peripheral blood from normal human donor drawn in CPD AS-1
    Using an RC-3B Centrifuge, spin a 450 ml bag of blood upright with no brake, 25 °C, 1,665 x g, for 7 min. Takes approximately 30 min to come to a stop without the brake.

  2. Assemble elutriator and chambers
    See Beckman JE6B Elutriation System and Rotor Assembly Manual.

  3. Sterilize and prepare elutriation chamber
    1. Attach 1 ml pipet to elutriation pump tubing and transfer tubing (by submerging in each solution) to each of the following in sequence:
      1. Wash #1 10% fresh bleach (in sterile milliQ H2O)
      2. Wash #2 300-500 ml: sterile milliQ H2O
      3. Wash #3 300-500 ml: 70% EtOH (sterile milliQ H2O)
      4. Spin elutriator rotor by hand for 30 sec to remove air pockets.
      5. Wash #4 300-500 ml: sterile milliQ H2O
      6. Wash #5 300-500 ml: sterile PBS
    2. Finally, equilibrate tubing and chamber with elutriation buffer (with 100-200 ml approximately 11-12 ml/min.flow rate).

  4. Collect blood from centrifuge
    1. Sit blood bag on extruder carefully and place onto the hooks on extruder.
    2. Wipe tube of blood bag and scissors with alcohol wipes to sterilize. Wipe tubing end with alcohol, attach clamp, and insert into a 250 ml plasma tube (if collecting plasma for later use) or waste (if not collecting plasma).
    3. With clamp shut on tubing, release press to squeeze bag gently.
    4. Open clamp to control flow to a fast drip/slow stream. Stop plasma collection when plasma is approximately 1 inch from top of bag.
    5. Transfer blood bag tube to T75 flask containing 50 ml PBS 0.02 mM EDTA, where you will collect approximately 25 ml plasma and 20 ml erythrocytes plus white cells) often called the buffy coat. Swirl flask gently. Total volume in flask should be approximately 90-100 ml.
    6. Tilt 50 ml tube containing 14-18 ml LSM and using a 10 ml pipette, layer approximately 30 ml buffy coat-PBS-EDTA carefully from flask onto LSM, repeat with second and third LSM tube.
    7. Remainder of buffy coat-PBS-EDTA should be layered onto last LSM tube.
    8. Balance LSM tubes, spin in RT6000 at 350 x g for 40 min, 22 °C with brake off.

  5. Blood/cells
    1. Remove diluted Plasma from top of tubes of LSM (approximately 25 ml).


      Figure 1. Adapted from http://www.genec.cl/stock/Mediatech.html

    2. Remove peripheral blood mononuclear cells (PMBC) interfaces, add to 40 ml PBS/1 mM EDTA in 50 ml tubes and invert to mix.
    3. Spin in RT6000 to remove platelets, 200 x g for 10 min. Pellet should be visible.
    4. Pour off most of the supernatant.
    5. Resuspend all pellets, pooling in a total of 8–10 ml elutriation buffer. Avoid air bubbles.
    6. Count PBMCs: should be between 7 x 108 and 1.5 x 109 total cells depending on the donor.

  6. Elutriation
    1. Run some elutriation buffer through to equilibrate (100 ml).
    2. Spin rotor manually for approximately 30 sec to remove air pockets.
    3. Close lid and allow speed to get up to 1,450 x g (final speed) before checking flow rate: should be between 11.4 and 11.6 ml/min.
    4. Transfer pipette attached to pump tubing into cells and pump full volume of cells (8-10 ml) before transferring pipette back to elutriation buffer. Avoid taking up any air by pinching tubing during transfer.
    5. Collect the effluent. Collect the first two 50 ml tubes of effluent which will contain the lymphocyte population (with some contaminating platelets and red cells) and spin down at 100 x g to remove platelets. Lyse any remaining erythrocytes in ACK at room temperature (by resuspending the pellet in the ACK and gently inverting the tube 1-2 times), wash the lymphocytes once in HBSS by resuspending in 25 ml, count cell concentration, pellet cells again at 100 x g, and resuspend the lymphocytes in complete media containing 50 U/ml IL-2 at 2 million/ml in complete media in T75 flasks. Monocytes will remain in chamber, due to size and density and centrifugation conditions.
    6. After 35 min run at 11.5 ml/min increase speed to 12.5 ml per min. Continue to run for 25 min (total runtime is 60 min).

  7. End of run
    1. Clamp output and input tubes before pump is turned off, then immediately stop centrifuge.
    2. Open lid and unlock black lock, then remove input tube first, then lower outlet tube.
    3. Remove elute chamber, with spout up.
    4. In hood, using a P1000 remove the 6.3 ml of sample from the elutriation chamber and add to 50 ml tube.
    5. Bring up volume up to 20 ml with 1x PBS. Count cells. Monocytes should be approximately 10-20% of original PBMC count. Wash cell pellet with sterile PBS. 200 x g for 10 min. Resuspend pellet to 100 x 106 cells/ml.
    6. Plate monocytes in 10 cm petri dishes at 0.5 x 10 E6/ml (10 ml total/dish) in complete media containing 25 ng/ml M-CSF for derivation into macrophages.

  8. Clean elutriator
    1. Place elute chamber back into rotor and rotor back into centrifuge chamber attaching tubes to wash out/decontaminate rotor, tubing and chamber.
    2. Undo clamps and run through 300 ml 10% bleach, 300 ml autoclaved H2O, 300 ml 70% EtOH, 300 ml water again. Disassemble rotor, set in 70% ethanol for 30% and let air dry overnight.

Recipes

  1. Complete media
    RPMI
    10% defined FBS
    1% P/S
    1% L-Gln
  2. ACK buffer
    To 450 ml milliQ water, add :
    4.145 g Ammonium Chloride (annhydrous)
    0.5 g potassium bicarbonate
    18.6 mg disodium EDTA
    Adjust pH to 7.4
    Bring final volume to 500 ml with milliQ water
    Fliter sterilize (Using Stericup) and store at 4 °C.
  3. 1x sterile Elutriation Buffer, pH 7.3
    8.3 g NaCl
    5 g dextrose in 1 L MQ H2O
    10 mM Na2HPO4
    3 mM NaH2PO4.H2O
    100,000 units Pen/Strep
    0.625% Human Serum Albumin (HSA) from Talecris Therapeutics
  4. 5x Phosphate Buffered Saline (PBS for washing cells), pH 7.0-7.2
    9.51 g dibasic phosphate, anhydrous (diluted is 6.7 mM Na2HPO4)
    4.56 g monobasic phosphate (diluted is 3.3 mM Na H2PO4-H2O)
    85 g NaCl (diluted will be .14 M)
    Dissolve and bring to final volume of 2000 ml with MilliQ (LPS-free) water. Filter sterilize. Diluted at room temperature should be pH 7.0-7.2, and K = 14-16. Store at 4 °C.

Acknowledgments

The original protocol was published in Bobak et al. (1986). Further modification and development of these methods was supported in part by NIH AI-41090 and T32 AI 60573.

References

  1. Bobak, D. A., Frank, M. M. and Tenner, A. J. (1986). Characterization of C1q receptor expression on human phagocytic cells: effects of PDBu and fMLP. J Immunol 136(12): 4604-4610. 
  2. Lionetti, F., Hunt, S. and Valeri, C. (1980). Methods of cell separation. Plenum Publishing Corporation, New York.

材料和试剂

  1. 来自正常人供体的外周血(450ml)
  2. CPD AS-1采血袋(柠檬酸盐 - 磷酸盐 - 葡萄糖,腺嘌呤盐水-1)(Baxter)
  3. 1x PBS-2mM EDTA
  4. 无菌milliQ H sub2 O
  5. 淋巴细胞分离培养基(LSM)(MP Biomedicals,目录号:0850494)
  6. HBSS(Fisher Scientific,目录号:21023CV)
  7. 乙醇(70%,1升)
  8. 10%漂白剂(1升)
  9. 10%定义的FBS(Hyclone,目录号:SH30070.03)
  10. 1%P/S(Mediatech,CellGro ,目录号:30-002-CIRF)
  11. 1%L-Gln(Mediatech,CellGro ,目录号:25-005-CI)
  12. 重组人IL-2(Pepro Tech,目录号:200-02)
  13. 重组人M-CSF(Pepro Tech,目录号:300-25)
  14. 25%白蛋白(人),USP(Talecris Plasma Resources,目录号:13533-684-20)
  15. 完成媒体(见配方)
  16. 1×无菌PBS(从5x PBS pH7.0-7.2稀释1:5)(参见Recipes)
  17. 氯化铵钾(ACK)缓冲液(参见配方)
  18. 1x无菌Elutriation Buffer(参见配方)
  19. 5x磷酸盐缓冲盐水(见配方)

设备

  1. 挤出机
  2. T75瓶
  3. Nunc蓝帽50毫升聚丙烯管(重要的单核细胞粘在其他管子低产量)
  4. 管件夹具
  5. 剪刀
  6. P1000 Pipetteman
  7. 培养皿
  8. 250 ml血浆管
  9. 异丙醇擦拭巾,单独包装
  10. Beckman JE6B Elutriation System and Rotor(Beckman Coulter,型号:JE6B-IM-3)(1992年11月)**
  11. 离心机(Sorvall,型号:RT6000或RC-3B)
  12. Stericup(Fisher Scientific,目录号:SCGVU11RE)
    ** Beckman Coulter有更新的型号Avanti J-301或J-26S XP与Elutriation Systems,但我们没有这方面的经验。

程序

  1. 在CPD AS-1中接受来自正常人类供体的450ml外周血 使用RC-3B离心机,在没有制动的情况下,在25℃,1665×g下直立旋转450ml袋的血液7分钟。 大约30分钟即可停止,无需制动。

  2. 装配淘洗器和室
    参见Beckman JE6B淘析系统和转子装配手册。

  3. 消毒并准备淘析室
    1. 将1ml移液管连接到淘析泵管和传送管(通过浸没在每种溶液中)到以下顺序中的每一个:
      1. 洗涤#1 10%新鲜漂白剂(在无菌milliQ H 2 O中)
      2. 洗涤#2 300-500ml:无菌milliQ H 2 O
      3. 洗涤#3 300-500ml:70%EtOH(无菌milliQ H 2 O)
      4. 用手旋转淘洗转子30秒,以清除气泡
      5. 洗涤#4 300-500ml:无菌milliQ H 2 O
      6. 洗涤#5 300-500ml:无菌PBS
    2. 最后,用淘析缓冲液(用100-200ml,大约11-12ml/min流速)平衡管道和室。

  4. 从离心机收集血液
    1. 将血袋小心地放在挤出机上,放在挤出机的钩子上
    2. 擦拭管血液袋和剪刀与酒精擦拭消毒。 用酒精擦拭管道末端,将夹子和插入250ml血浆管(如果收集血浆用于以后使用)或浪费(如果不收集血浆)。
    3. 用夹子关闭管道,释放压力机轻轻挤压袋子
    4. 打开夹子控制流量到快速滴/慢流。 等离子体距离袋子顶部约1英寸时,停止等离子体收集。
    5. 转移血袋管到含有50 ml PBS 0.02 mM EDTA的T75瓶中,在那里你将收集约25 ml血浆和20 ml红细胞加白色细胞),通常称为血沉棕黄层。 轻轻旋转烧瓶。 烧瓶中的总体积应为约90-100ml。
    6. 倾斜50ml含有14-18ml LSM的管,并使用10ml移液管,从烧瓶小心地将约30ml血沉棕黄层-PBS-EDTA层到LSM上,用第二和第三LSM管重复。
    7. 剩余的血沉棕黄层-PBS-EDTA应该铺在最后的LSM管上
    8. 平衡LSM管,在350℃下在RT6000中旋转40分钟,在制动关闭的情况下在22℃下旋转。
  5. 血细胞
    1. 从LSM管(约25 ml)的顶部取出稀释的血浆

      图1.改编自 http://www.genec.cl/stock/Mediatech.html

    2. 去除外周血单核细胞(PMBC)接口,加入到50ml管中的40ml PBS/1mM EDTA中,并倒转混合。
    3. 在RT6000中旋转以去除血小板,200×g/10分钟。 颗粒应可见。
    4. 倒出大部分上清液。
    5. 重悬所有颗粒,在总共8-10ml淘析缓冲液中汇集。 避免气泡。
    6. 计数PBMC:根据供体,应当在7×10 8个和1.5×10 9个总细胞之间。

  6. 淘洗
    1. 运行一些淘析缓冲液通过平衡(100 ml)。
    2. 手动旋转转子约30秒,以清除气泡。
    3. 在检查流速之前,关闭盖子并使速度达到1,450×g(最终速度):应该在11.4和11.6ml/min之间。
    4. 将连接到泵管的移液管移入细胞,并且在将移液管移回淘析缓冲液之前泵送全部体积的细胞(8-10ml)。 避免在转移过程中夹住管道吸收任何空气。
    5. 收集流出物。 收集前两个50ml的流出物管,其将含有淋巴细胞群(具有一些污染的血小板和红细胞)并以100×g离心至 去除血小板。 在室温下裂解ACK中的任何剩余的红细胞(通过将沉淀重悬在ACK中并轻轻颠倒管1-2次),在HBSS中洗涤淋巴细胞一次,通过重悬于25ml,计数细胞浓度, 并将淋巴细胞在含有50U/ml IL-2的完全培养基中以2百万/ml在T75烧瓶中的完全培养基中重悬。 由于大小和密度和离心条件,单核细胞将保留在室中。
    6. 在35分钟后以11.5ml/min运行,将速度增加至12.5ml/min。 继续运行25分钟(总运行时间为60分钟)。

  7. 运行结束
    1. 在泵关闭之前夹住输出和输入管,然后立即停止离心
    2. 打开盖子并解锁黑色锁,然后先取下输入管,然后放下出口管。
    3. 取出洗脱室,喷出。
    4. 在罩中,使用P1000从淘析室中取出6.3ml样品并加入到50ml管中。
    5. 用1x PBS将体积增加至20 ml。 计数单元格。 单核细胞应该是原始PBMC计数的约10-20%。 用无菌PBS洗涤细胞沉淀。 200 x g 10分钟。 将沉淀重悬浮至100×10 6个细胞/ml
    6. 在含有25ng/ml M-CSF的完全培养基中,以0.5×10 6个/ml(总共10ml /皿)在10cm培养皿中平板单核细胞,用于衍生成巨噬细胞。
  8. 清洁淘汰
    1. 将洗脱室放回转子和转子回到离心机室连接管,以洗涤/净化转子,管道和室。
    2. 松开夹子并再次通过300ml 10%漂白剂,300ml高压灭菌的H 2 O,300ml 70%EtOH,300ml水。 拆卸转子,置于70%乙醇中30%,让空气干燥过夜

食谱

  1. 填写媒体
    RPMI
    10%定义的FBS
    1%P/S
    1%L-Gln
  2. ACK缓冲区
    向450ml milliQ水中加入:
    4.145克氯化铵(无水)
    0.5克碳酸氢钠 18.6mg EDTA二钠
    将pH调节至7.4
    用milliQ水
    使最终体积达到500 ml Fliter灭菌(使用Stericup)并储存在4°C
  3. 1x无菌Elutriation Buffer,pH 7.3
    8.3克NaCl
    5g在1L MQ H 2 O中的葡萄糖 10mM Na 2 HPO 4
    3mM NaH 2 PO 4 subO 2·H 2 O 2·h / 100,000单位Pen/Strep
    0.625%来自Talecris Therapeutics的人血清白蛋白(HSA)
  4. 5x磷酸盐缓冲盐水(用于洗涤细胞的PBS),pH 7.0-7.2
    9.51g无水磷酸氢二铵(稀释为6.7mM Na 2 HPO 4)
    4.56g磷酸二氢盐(稀释为3.3mM Na 2 H 2 PO 4·H 2 O)
    85g NaCl(稀释为0.14M)
    溶解并使用MilliQ(不含LPS)水达到2000ml的最终体积。 过滤灭菌。 在室温下稀释应为pH 7.0-7.2,K = 14-16。 储存于4°C。

致谢

原始协议在Bobak等人(1986)中公开。 这些方法的进一步修改和开发部分由NIH AI-41090和T32 AI 60573支持。

参考文献

  1. Bobak,D.A.,Frank,M.M。和Tenner,A.J。(1986)。 人吞噬细胞上C1q受体表达的表征:PDBu和fMLP的作用。 em> J Immunol 136(12):4604-4610。 
  2. Lionetti,F.,Hunt,S.and Valeri,C。(1980)。 细胞分离方法。Plenum Publishing Corporation,New York。
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How to cite this protocol: Clarke, E. V., Benoit, M. E. and Tenner, A. J. (2013). Purification of Human Monocytes and Lymphocyte Populations by Counter Current Elutriation – A Short Protocol. Bio-protocol 3(23): e981. DOI: 10.21769/BioProtoc.981; Full Text



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