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Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC)
肿瘤相关巨噬细胞(TAM)和肿瘤相关树突细胞(TADC)的纯化   

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Abstract

Tumors are heterogeneous microenvironments where complex interactions take place between neoplastic cells and infiltrating inflammatory cells, such as tumor-associated macrophages (TAM) and tumor-associated dendritic cells (TADC). The relevance of tumor-infiltrating mononuclear myeloid cells is underscored by clinical studies showing a correlation between their abundance and poor prognosis (Laoui et al., 2011). These cells are able to promote tumor progression via several mechanisms, including induction of angiogenesis, remodeling of the extracellular matrix, stimulation of cancer cell proliferation and metastasis and the inhibition of adaptive immunity (Laoui et al., 2011). Moreover, mononuclear myeloid cells are characterized by plasticity and versatility in response to microenvironmental signals, resulting in different activation states, as illustrated by the presence of distinct functional TAM subsets in tumors (Movahedi et al., 2010; Laoui et al., 2014). Here, we describe a valuable isolation technique for TAM and TADC permitting their molecular and functional characterization.

Keywords: Tumor-associated macrophages(肿瘤相关巨噬细胞), Tumor-associated dendritic cells(树突细胞肿瘤相关), Tumor single cell suspension(肿瘤单细胞悬液), Macrophage purification(巨噬细胞的分离纯化), Dendritic cell purification(树突细胞的纯化)

Materials and Reagents

  1. Mouse
  2. RPMI-1640 medium (RPMI) (Life Technologies, catalog number: 52400-041 )
  3. Collagenase I (Worthington Biochemical, catalog number: LS004214 )
  4. Collagenase IV (Worthington Biochemical, catalog number: LS004209 )
  5. DNase I (Worthington Biochemical, catalog number: LS002060 )
  6. Hank’s buffered salt solution (HBSS) (Life Technologies, Gibco®)
  7. NH4Cl (Merck KGaA)
  8. KHCO3 (Merck KGaA)
  9. EDTA (Duchefa Biochemie)
  10. Fetal calf serum (FCS) (Life Technologies, Gibco®)
  11. Lymphoprep (Axis-shield, catalog number: 1114547 )
  12. LS colums (Miltenyi Biotec, catalog number: 130-042-401 )
  13. Anti-CD11b microbeads (Miltenyi Biotec, catalog number: 130-049-601 )
  14. Anti-CD11c microbeads (Miltenyi Biotec, catalog number: 130-052-001 )
  15. Purified CD16/CD32 (FcBlock) (clone 2.4G2) (BD Biosciences, catalog number: 553142 )
  16. PE-Cy7-conjugated anti-CD11b antibody (clone M1/70) (BD Biosciences, catalog number: 552850 )
  17. AF647-conjugated anti-Ly6C antibody (clone ER-MP20) (Bio-Rad Laboratories, AbD Serotec®, catalog number: MCA2389A647 )
  18. PerCP-Cy5.5-conjugated anti-I-A/I-E (MHC-II) antibody (clone M5/114.15.2) (BioLegend, catalog number: 107626 )
  19. FITC-conjugated anti-Ly6G antibody (clone 1A8) (BD Biosciences, catalog number: 551460 )
  20. PE-conjugated anti-SiglecF antibody (clone E50-2440) (BD Biosciences, catalog number: 552126 )
  21. PE-conjugated anti-CD11c antibody (clone HL3) (BD Biosciences, catalog number: 553802 )
  22. Tumor digestion medium (see Recipes)
  23. Erythrocyte lysis buffer (see Recipes)
  24. MACS buffer (see Recipes)
  25. Sorting buffer (see Recipes)
  26. Complete medium (see Recipes)

Equipment

  1. Polyester filters cut in 10 x 10 cm squares (thread diameter 70 μm) (Spectrum® Laboratories, catalog number: 146490 )
  2. 6-well plates (Greiner Bio-One GmbH, catalog number: 657185 )
  3. 10 ml syringes (Omnifix, catalog number: 473203 )
  4. BD Falcon 50 ml polypropylene tubes (BD Biosciences, catalog number: 2070 )
  5. BD Falcon 15 ml polypropylene tubes (BD Biosciences, catalog number: 2096 )
  6. BD Falcon 5 ml polypropylene round-bottom tube (BD Biosciences, catalog number: 352063 )
  7. Sterile culture hood
  8. Surgical scissors and forceps
  9. 37 °C, 5% CO2 cell culture incubator
  10. Pipettes
  11. Centrifuges
  12. Shaker
  13. Microscope
  14. MidiMACSTM Separator and MultiStand (Miltenyi Biotec, catalog number: 130-042-301 )
  15. Multicolour FACS sorter (BD Biosciences, Aria flow cytometer)

Procedure

  1. Preparation of a tumor single cell suspension
    1. Sacrifice the mouse when the tumor has reached the desired diameter (as from the moment a tumor becomes palpable, it can be analyzed. The maximal tumor diameter depends on the limits set by the ethical commission and may vary in different countries) and restrain it by pinning its paws into a foam surface using syringe needles. Make a parallel incision from the base of the tail up to the neck along the mouse’s abdomen and to the paws without puncturing the peritoneum. Gently pull back the skin and pin it to the foam surface to reveal the tumor. Here, LLC lung carcinoma was used as an example, however the protocol is applicable for all solid tumors (Figure 1A).
    2. Cut the tumor free from the skin and the body. Try to remove as much excess tissue surrounding the tumor as possible and take care to exclude the draining lymph node (Figure 1A).
    3. Store the harvested tumors in 2 ml RMPI medium in a 6-well plate on ice until the digestion procedure (Figure 1B).
    4. Cut the tumors in small pieces (1-1.5 mm) using scissors or a scalpel, subsequently add 1 ml digestion medium in the same well and incubate subsequently at 37 °C for 25 min (Figure 1C).
    5. Crush the tumors with the plunger of 10 ml syringe, add 5 ml RPMI medium and homogenize by thoroughly pipetting with a 10 ml pipet.
    6. Filter the tumor suspensions through a 70 µm sterile nylon gauze into a sterile 50 ml conical tube and wash the gauze with 10 ml RPMI medium.
    7. Centrifuge the 50 ml tubes at 450 x g for 6 min at 4 °C and discard the supernatants.
    8. Remove the red blood cells by resuspending the pellet in 4 ml erythrocyte lysis buffer and leave at room temperature for 2 min.
    9. Neutralize by adding 12 ml RPMI medium, and transfer the suspension to a new 50 ml tube through a 70 µm sterile nylon gauze.
    10. Centrifuge the 50 ml tubes at 450 x g for 6 min at 4 °C and discard the supernatants.
    11. Count the living cells using trypan bleu and resuspend the cell pellet in Lymphoprep at a concentration of approximately 1-2 x 107 cells/ml and transfer the suspension to sterile 15 ml tube(s) (6 ml/tube). Cover this cell suspension slowly and cautiously with 6 ml RPMI to obtain a two-phase gradient (Figure 1D).
    12. Centrifuge the gradients at 800 x g for 30 min at room temperature without acceleration or break.
    13. Carefully collect the interphase and the upper phase containing the RPMI medium and as less Lymphoprep contamination as possible (Figure 1E). The interphase contains the living cells and is enriched in myeloid cells, and transfer to a new sterile 15 ml tube. Wash by filling the 15 ml tube unto the top with MACS buffer, centrifuge at 800 x g for 5 min at 4 °C and discard the supernatants.
    14. Resuspend the cells in MACS buffer at a concentration of 108 cells/ml.


      Figure 1. Tumor single-cell preparation. A. LLC tumor-bearing mouse. The arrow points to the draining lymph node, in this case the inguinal lymph node. B. Tumors in a 6 well plate in 2 ml RPMI medium. C. Tumors after digestion. D. Prepared gradient. E. Gradient after centrifugation with a clearly visible interphase.

  2. Purification of tumor-associated macrophages (TAM)
    1. Add a 5 µl aliquot of anti-CD11b magnetic microbeads per 107 cells and incubate for 20 min at 4 °C on an orbital shaker at 50 rpm.
    2. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    3. Place an LS column in a MidiMACS Separator attached to a magnetic MultiStand and wash it by putting 3 ml MACS buffer on the top. The liquid passes the column by gravity.
    4. Resuspend the pelleted cells in 1 ml MACS buffer and pipette the labeled cell suspension on top of the LS separation column. Wash the column by adding 3 x 3 ml MACS buffer.
    5. Remove the LS column from the separator and wash the magnetically labeled cells out with 5 ml MACS buffer using a plunger in a sterile 15 ml tube.
    6. Incubate the CD11b+ cell suspension with rat anti-mouse CD16/CD32 (10 µg per 107 cells) on ice water for 20 min, in order to block the Fc receptors present on the cells’ surface.
    7. Incubate the cell suspension with fluorescently labeled antibodies (1 µg per 107 cells) for another 20 min on ice water, protected from exposure to light. TAM can be sorted on a BD FACS Aria as CD11bpos Ly6Gneg SiglecFneg Ly6Clow cells and can be separated into MHC-IIhigh and MHC-IIlow TAM subsets (Laoui et al., 2014) (Figure 2A).
    8. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    9. Meanwhile precoat 5 ml polypropylene round-bottom tubes and 15 ml tubes with heat-inactivated fetal calf serum, add respectively 1 ml or 2 ml heat-inactivated fetal calf serum. Shake the tubes gently by hand so that the heat-inactivated fetal calf serum covers the whole surface of the tube, and discard the excess of heat-inactivated fetal calf serum. This will prevent the cells to stick to the tubes and hence enhance the recovery of cells.
    10. Resuspend the pellet in 1 ml sorting buffer per 107 cells and transfer into a sterile 5 ml polypropylene round-bottom tube precoated with heat-inactivated fetal calf serum.
    11. Collect the sorted TAM in 15 ml tubes precoated with heat-inactivated fetal calf serum containing 3 ml complete medium.

  3. Purification tumor-associated dendritic cells (TADC)
    1. Add a 5 µl aliquot of anti-CD11c microbeads per 107 cells and incubate for 20 min at 4 °C on an orbital shaker at 50 rpm.
    2. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    3. Place an LS column in a MidiMACS Separator attached to a magnetic MultiStand and wash it by putting 3 ml MACS buffer on the top. The liquid passes the column by gravity.
    4. Resuspend the pelleted cells in 1 ml MACS buffer and pipette the labeled cell suspension on top of the LS separation column. Wash the column by adding 3 x 3 ml MACS buffer.
    5. Remove the LS column from the separator and wash the magnetically labeled cells out with 5 ml MACS buffer using a plunger in a sterile 15 ml tube.
    6. Incubate the CD11c+ cell suspension with rat anti-mouse CD16/CD32 (10 µg per 107 cells) on ice water for 20 min, in order to block the Fc receptors present on the cells’ surface.
    7. Incubate the cell suspension with fluorescently labeled antibodies (1 µg per 107 cells) for another 20 min on ice water, protected from exposure to light. TADC can be sorted on a BD FACS Aria as Ly6Gneg SiglecFneg CD11cpos MHC-IIpos cells. Take care to gate only on the MHC-IIhighest cells and to exclude the MHC-IIhigh TAM, which also express CD11c, albeit at a lower level than TADC (Figure 2B).
    8. Wash by adding 10 ml MACS buffer, centrifuge at 450 x g for 6 min at 4 °C and discard the supernatants.
    9. Meanwhile precoat 5 ml polypropylene round-bottom tubes and 15 ml tubes with heat-inactivated fetal calf serum, add respectively 1 ml or 2 ml heat-inactivated fetal calf serum. Shake the tubes gently by hand so that the heat-inactivated fetal calf serum covers the whole surface of the tube, and discard the excess of heat-inactivated fetal calf serum. This will prevent the cells to stick to the tubes and hence enhance the recovery of cells.
    10. Resuspend the pellet in 1 ml sorting buffer per 107 cells and transfer into a sterile 5 ml polypropylene round-bottom tube precoated with heat-inactivated fetal calf serum.
    11. Collect the sorted TADC in 15 ml tubes precoated with heat-inactivated fetal calf serum containing 3 ml complete medium.


      Figure 2. TAM and TADC gating. A. TAM subsets can be gated using MHC-II and Ly6C in CD11bpos Ly6Gneg SiglecFneg tumor single-cell suspensions. B. TADC can be gated using MHC-II and CD11c in Ly6Gneg SiglecFneg tumor single-cell suspensions.

Recipes

  1. Tumor digestion medium
    10 U/ml Collagenase I
    400 U/ml Collagenase IV
    30 U/ml DNAse I
    All diluted in HBSS
    Aliquoted and frozen at -20 °C
  2. Erythrocyte lysis buffer
    8.29 g/L NH4Cl
    1 g/L KHCO3
    37.2 mg/L EDTA
    Bring at pH 7.2
  3. MACS buffer
    Hank’s buffered salt solution
    0.5% (v/v) heat-inactivated fetal calf serum
    2 mM EDTA
  4. Sorting buffer
    Hank’s buffered salt solution
    0.5% (v/v) heat-inactivated FCS
    5 mM EDTA
  5. Complete medium
    Roswell Park Memorial Institute (RPMI)-1640
    10% (v/v) heat-inactivated fetal calf serum (FCS)
    300 μg/ml L-glutamine
    100 U/ml penicillin
    100 μg/ml streptomycin
    0.02 mM β-mercaptoethanol
    1 mM sodium pyruvate
    1 mM non-essential amino acids

Acknowledgments

The authors thank FWO-Vlaanderen, the “Stichting tegen Kanker” and the “Vlaamse Liga tegen Kanker” for their support.

References

  1. Laoui, D., Van Overmeire, E., Di Conza, G., Aldeni, C., Keirsse, J., Morias, Y., Movahedi, K., Houbracken, I., Schouppe, E. and Elkrim, Y. (2014). Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population. Cancer Res 74(1): 24-30.
  2. Laoui, D., Van Overmeire, E., Movahedi, K., Van den Bossche, J., Schouppe, E., Mommer, C., Nikolaou, A., Morias, Y., De Baetselier, P. and Van Ginderachter, J. A. (2011). Mononuclear phagocyte heterogeneity in cancer: different subsets and activation states reaching out at the tumor site. Immunobiology 216(11): 1192-1202.
  3. Movahedi, K., Laoui, D., Gysemans, C., Baeten, M., Stange, G., Van den Bossche, J., Mack, M., Pipeleers, D., In't Veld, P., De Baetselier, P. and Van Ginderachter, J. A. (2010). Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes. Cancer Res 70(14): 5728-5739.

简介

肿瘤是异质微环境,其中在肿瘤细胞和浸润性炎症细胞例如肿瘤相关巨噬细胞(TAM)和肿瘤相关树突细胞(TADC)之间发生复杂的相互作用。临床研究强调了肿瘤浸润性单核骨髓细胞的相关性,显示其丰度与不良预后之间的相关性(Laou等人,2011)。这些细胞能够通过几种机制促进肿瘤进展,包括诱导血管生成,重塑细胞外基质,刺激癌细胞增殖和转移以及抑制适应性免疫(Laoui等人,2011 )。此外,单核骨髓细胞的特征在于响应于微环境信号的可塑性和通用性,导致不同的活化状态,如通过在肿瘤中存在不同的功能性TAM亚型所说明的(Movahedi等人,2010; Laoui et al。,2014)。在这里,我们描述了允许其分子和功能表征的TAM和TADC的有价值的分离技术。

关键字:肿瘤相关巨噬细胞, 树突细胞肿瘤相关, 肿瘤单细胞悬液, 巨噬细胞的分离纯化, 树突细胞的纯化

材料和试剂

  1. 鼠标
  2. RPMI-1640培养基(RPMI)(Life Technologies,目录号:52400-041)
  3. 胶原酶I(Worthington Biochemical,目录号:LS004214)
  4. 胶原酶IV(Worthington Biochemical,目录号:LS004209)
  5. DNase I(Worthington Biochemical,目录号:LS002060)
  6. Hank's缓冲盐溶液(HBSS)(Life Technologies,Gibco )
  7. NH 4 Cl(Merck KGaA)
  8. (Merck KGaA)
  9. EDTA(Duchefa Biochemie)
  10. 胎牛血清(FCS)(Life Technologies,Gibco )
  11. Lymphoprep(Axis-shield,目录号:1114547)
  12. LS colums(Miltenyi Biotec,目录号:130-042-401)
  13. 抗CD11b微珠(Miltenyi Biotec,目录号:130-049-601)
  14. 抗CD11c微珠(Miltenyi Biotec,目录号:130-052-001)
  15. 纯化的CD16/CD32(FcBlock)(克隆2.4G2)(BD Biosciences,目录号:553142)
  16. PE-Cy7缀合的抗CD11b抗体(克隆M1/70)(BD Biosciences,目录号:552850)
  17. AF647缀合的抗Ly6C抗体(克隆ER-MP20)(Bio-Rad Laboratories,AbD Serotec ,目录号:MCA2389A647)
  18. PerCP-Cy5.5-结合的抗I-A/I-E(MHC-II)抗体(克隆M5/114.15.2)(BioLegend,目录号:107626)
  19. FITC缀合的抗Ly6G抗体(克隆1A8)(BD Biosciences,目录号:551460)
  20. PE缀合的抗SiglecF抗体(克隆E50-2440)(BD Biosciences,目录号:552126)
  21. PE缀合的抗CD11c抗体(克隆HL3)(BD Biosciences,目录号:553802)
  22. 肿瘤消化介质(见配方)
  23. 红细胞裂解缓冲液(参见配方)
  24. MACS缓冲区(参见配方)
  25. 排序缓冲区(请参阅配方)
  26. 完整介质(见配方)

设备

  1. 在10×10cm正方形(螺纹直径70μm)(Spectrum 实验室,目录号:146490)中切割的聚酯过滤器
  2. 6孔板(Greiner Bio-One GmbH,目录号:657185)
  3. 10ml注射器(Omnifix,目录号:473203)
  4. BD Falcon 50ml聚丙烯管(BD Biosciences,目录号:2070)
  5. BD Falcon 15ml聚丙烯管(BD Biosciences,目录号:2096)
  6. BD Falcon 5ml聚丙烯圆底管(BD Biosciences,目录号:352063)
  7. 无菌培养罩
  8. 外科剪刀和镊子
  9. 37℃,5%CO 2细胞培养箱中培养
  10. 移液器
  11. 离心机
  12. 振动器
  13. 显微镜
  14. MidiMACS TM Separator and MultiStand(Miltenyi Biotec,目录号:130-042-301)
  15. 多色FACS分选仪(BD Biosciences,Aria流式细胞仪)

程序

  1. 制备肿瘤单细胞悬液
    1. 牺牲 当肿瘤已达到所需直径时(如从 瞬间肿瘤变得明显,可以分析。 最大肿瘤 直径取决于道德委员会设定的限制,可能 在不同的国家变化),并通过把它的爪子限制在一个 泡沫表面使用注射器针。 从平行切口 基部的尾巴沿着鼠标的腹部和颈部的颈部 爪不刺穿腹膜。 轻轻拉回皮肤和 将其固定到泡沫表面以显露肿瘤。 这里,LLC肺癌   被用作示例,然而该协议适用于所有 实体瘤(图1A)。
    2. 切除肿瘤从皮肤和 身体。 尝试删除尽可能多的肿瘤周围的组织 可能并注意排除引流淋巴结(图1A)。
    3. 将收获的肿瘤存储在2ml RMPI培养基中的6孔板在冰上,直到消化程序(图1B)。
    4. 使用剪刀或a。切割小块(1-1.5毫米)肿瘤 解剖刀,随后在相同的孔中加入1ml消化培养基 随后在37℃孵育25分钟(图1C)
    5. 粉碎 肿瘤用柱塞注射10ml注射器,加入5ml RPMI培养基 通过用10ml移液管彻底吹打匀化。
    6. 过滤 将肿瘤悬浮通过70μm无菌尼龙纱布进入无菌   50ml锥形管,并用10ml RPMI培养基洗涤纱布
    7. 在4℃下以450xg离心50分钟的管子6分钟,弃去上清液。
    8. 通过将沉淀重悬在4ml中来除去红细胞 红细胞裂解缓冲液,并在室温下放置2分钟。
    9. 通过加入12ml RPMI培养基中和,并转移悬浮液 到通过70μm无菌尼龙纱布的新的50ml管。
    10. 在4℃下以450xg离心50分钟的管子6分钟,弃去上清液。
    11. 使用台盼蓝计数活细胞并重悬细胞 沉淀在Lymphoprep中,浓度为约1-2×10 7个细胞/ml,并将悬浮液转移到无菌的15ml管(6) ml /管)。用6 ml缓慢小心地覆盖此细胞悬液 RPMI获得两相梯度(图1D)。
    12. 在室温下以800×g离心梯度30分钟,不加速或断裂。
    13. 仔细收集中间相和上相含有  RPMI培养基和尽可能少的Lymphoprep污染(图 1E)。间期包含活细胞并富含骨髓  细胞,并转移到新的无菌15ml管中。通过填充洗涤15  ml管,用MACS缓冲液至上部,以800×g离心5分钟 在4℃并弃去上清液
    14. 将细胞以10 8细胞/ml的浓度重悬于MACS缓冲液中

      图1.肿瘤单细胞制备。 A。 LLC荷瘤小鼠。 箭头指向引流淋巴结,在这种情况下是腹股沟 淋巴结。 B.在6孔板中的肿瘤在2ml RPMI培养基中。 C.肿瘤 消化后。 D.制备梯度。 E.离心后的梯度 具有清晰可见的界面。

  2. 肿瘤相关巨噬细胞(TAM)的纯化
    1. 加入5μl等份的抗CD11b磁性微珠/10 10个细胞 并在轨道摇床上以50rpm在4℃下孵育20分钟
    2. 通过加入10ml MACS缓冲液洗涤,在4℃下以450xg离心6分钟,弃去上清液。
    3. 将LS柱放在连接到磁性的MidiMACS分离器中 MultiStand并通过将3ml MACS缓冲液放在顶部来洗涤。 的 液体在重力作用下通过色谱柱
    4. 重悬沉淀的细胞   在1ml MACS缓冲液中,并在其上吸取标记的细胞悬浮液 LS分离柱。 通过加入3×3ml MACS洗涤柱 缓冲区
    5. 从分离器中取出LS柱并洗涤 磁性标记的细胞用5ml MACS缓冲液,使用柱塞   无菌15 ml管
    6. 与大鼠孵育CD11b +细胞悬浮液  抗 - 小鼠CD16/CD32(10μg/10 7个细胞)在冰水上孵育20分钟,在 以阻断存在于细胞表面上的Fc受体
    7. 孵育细胞悬液与荧光标记的抗体(1微克  每10个细胞)在冰水上再保持20分钟,防止暴露  光。 TAM可以在BD FACS Aria上作为CD11b pos Ly6G neg SiglecF neg Ly6C低级细胞进行分选,分为MHC-II 和 MHC-II TAM亚群(Laou等人,2014)(图2A)。
    8. 通过加入10ml MACS缓冲液洗涤,在4℃下以450xg离心6分钟并弃去上清液。
    9. 同时预涂5毫升聚丙烯圆底管和15毫升 管用热灭活的胎牛血清,分别加入1ml或2μl  ml热灭活的胎牛血清。用手轻轻摇动试管  热灭活的胎牛血清覆盖整个表面 管,并丢弃过量的热灭活的胎牛血清。 这将防止细胞粘在管上,从而增强 恢复细胞。
    10. 将沉淀重悬在1ml分选缓冲液中 每10 7个细胞并转移到无菌的5ml聚丙烯中 用热灭活的胎牛血清预涂的圆底管
    11. 收集排序TAM在15毫升管预热包含3毫升完全培养基的热灭活的胎牛血清。

  3. 纯化肿瘤相关树突状细胞(TADC)
    1. 每10 7个细胞加入5μl抗CD11c微珠的等分试样,并在轨道摇床上在50rpm下在4℃孵育20分钟。
    2. 通过加入10ml MACS缓冲液洗涤,在4℃下以450xg离心6分钟,弃去上清液。
    3. 将LS柱放在连接到磁性的MidiMACS分离器中 MultiStand并通过将3ml MACS缓冲液放在顶部来洗涤。 的 液体在重力作用下通过色谱柱
    4. 重悬沉淀的细胞   在1ml MACS缓冲液中,并在其上吸取标记的细胞悬浮液 LS分离柱。 通过加入3×3ml MACS洗涤柱 缓冲区
    5. 从分离器中取出LS柱并洗涤 磁性标记的细胞用5ml MACS缓冲液,使用柱塞   无菌15 ml管
    6. 将CD11c +细胞悬浮液与大鼠孵育  抗 - 小鼠CD16/CD32(10μg/10 7个细胞)在冰水上孵育20分钟,在 以阻断存在于细胞表面上的Fc受体
    7. 孵育细胞悬液与荧光标记的抗体(1微克  每10个细胞)在冰水上再保持20分钟,防止暴露  光。 TADC可以在BD FACS Aria上作为Ly6G neg SiglecF neg CD11c pos MHC-II pos 细胞进行分选。注意仅在MHC-II 最高细胞上门而排除也表达CD11c的MHC-II TAM, 虽然水平低于TADC(图2B)。
    8. 通过加入10ml MACS缓冲液洗涤,在4℃下以450xg离心6分钟,弃去上清液。
    9. 同时预涂5毫升聚丙烯圆底管和15毫升 管用热灭活的胎牛血清,分别加入1ml或2μl ml热灭活的胎牛血清。用手轻轻摇动试管  热灭活的胎牛血清覆盖整个表面 管,并丢弃过量的热灭活的胎牛血清。 这将防止细胞粘在管上,从而增强 恢复细胞。
    10. 将沉淀重悬在1ml分选缓冲液中 每10 7个细胞并转移到无菌的5ml聚丙烯中 用热灭活的胎牛血清预包被的圆底管。
    11. 收集排序TADC在15毫升管预热包含3毫升完全培养基的热灭活的胎牛血清。


      图2. TAM和TADC门控。A. TAM子集可以使用MHC-II门控  和Ly6C在CD11b pos Ly6G neg SiglecF neg肿瘤单细胞悬浮液中。 B.TADC可以使用在Ly6G阴性肿瘤中的MHC-II和CD11c门控 单细胞悬液。

食谱

  1. 肿瘤消化培养基
    10 U/ml胶原酶I
    400U/ml胶原酶IV
    30U/ml DNAse I
    所有稀释在HBSS中
    分装并在-20°C下冷冻
  2. 红细胞裂解缓冲液
    8.29g/L NH 4 Cl/h 1 g/L KHCO 3
    37.2mg/L EDTA
    在pH 7.2下冲洗
  3. MACS缓冲区
    汉克缓冲盐溶液
    0.5%(v/v)热灭活的胎牛血清 2mM EDTA
  4. 排序缓冲区
    汉克缓冲盐溶液
    0.5%(v/v)热灭活的FCS 5 mM EDTA
  5. 完成媒介
    罗斯威尔公园纪念研究所(RPMI)-1640
    10%(v/v)热灭活的胎牛血清(FCS)
    300μg/ml L-谷氨酰胺 100 U/ml青霉素
    100μg/ml链霉素 0.02mMβ-巯基乙醇 1mM丙酮酸钠 1 mM非必需氨基酸

致谢

作者感谢FWO-Vlaanderen,"Stichting tegen Kanker"和"Vlaamse Liga tegen Kanker"的支持。

参考文献

  1. Laoui,D.,Van Overmeire,E.,Di Conza,G.,Aldeni,C.,Keirsse,J.,Morias,Y.,Movahedi,K.,Houbracken,I.,Schouppe,E。和Elkrim,Y (2014)。 肿瘤缺氧不会驱动肿瘤相关巨噬细胞的分化,而是微调M2- like macrophage population。 Cancer Res 74(1):24-30。
  2. Laoui,D.,Van Overmeire,E.,Movahedi,K.,Van den Bossche,J.,Schouppe,E.,Mommer,C.,Nikolaou,A.,Morias,Y.,De Baetselier, Ginderachter,JA(2011)。 癌症中的单核吞噬细胞异质性:在肿瘤部位不同的亚群和激活状态。 Immunobiology 216(11):1192-1202
  3. Maohedi,K.,Laoui,D.,Gysemans,C.,Baeten,M.,Stange,G.,Van den Bossche,J.,Mack,M.,Pipeleers,D., De Baetselier,P。和Van Ginderachter,JA(2010)。 不同的肿瘤微环境包含源自Ly6C(高)单核细胞的巨噬细胞的功能不同亚群。 Cancer Res 70(14):5728-5739。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Laoui, D., Overmeire, E. V., Keirsse, J., Movahedi, K. and Ginderachter, J. A. (2014). Purification of Tumor-Associated Macrophages (TAM) and Tumor-Associated Dendritic Cells (TADC). Bio-protocol 4(22): e1294. DOI: 10.21769/BioProtoc.1294.
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Margarita Bartish
Karolinska Institutet
Hi!

I have a question with regards to your use of lymphoprep to purify the dissociated tumor: I am interested in collecting Ly6G+ cells from the tumor (putative granulocytic MDSCs). The lymphoprep description from the manufacturer (in my case, StemCell) states that "differences in cell density are exploited to separate granulocytes and erythrocytes from mononuclear cells", seemingly precluding its use for my purposes. But I see a Ly6G+ population in your facs plots. Does lymphoprep behave differently if it used on whole blood compared to as you do, dissociated tumor tissue? In your experience, based on the percentages of Ly6G+ you see, do you think it would be possible to extract a workable number of cells from a tumor sample? Thank you for your input!
11/23/2016 5:00:46 AM Reply