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Several studies have shown that the detrimental influence of abdominal obesity on metabolic processes is mediated by the intra-abdominal fat depot. Visceral adipose tissue has been shown to be an independent risk factor for coronary heart disease, hypertension, impaired glucose tolerance and Diabetes Mellitus Type 2 (DM2). Diet-induced obesity in mice, primarily of the C57BL/6J strain, is a commonly used method to study the development of insulin resistance as a model for DM2. The white or visceral adipose tissue (here referred to as VAT), especially the fat around the gonads, is a commonly used organ of study in this model, as it accumulates large numbers of lymphocytes in response to diet-induced obesity. The protocol below describes the isolation of lymphocytes from the stromal vascular fraction (SVF) from VAT.

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Isolation of Lymphocytes from Murine Visceral Adipose Tissue
从鼠内脏脂肪组织中分离淋巴细胞

免疫学 > 免疫细胞分离 > 淋巴细胞
作者: Sonja Valentić
Sonja ValentićAffiliation: Department of Histology & Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
Bio-protocol author page: a2752
Felix M. Wensveen
Felix M. WensveenAffiliation 1: Department of Histology & Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
Affiliation 2: Department of Experimental Immunology, Amsterdam Medical Centre, Amsterdam, The Netherlands
Bio-protocol author page: a2750
 and Bojan Polić
Bojan PolićAffiliation: Department of Histology & Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
For correspondence: bojan.polic@medri.uniri.hr
Bio-protocol author page: a2751
Vol 5, Iss 23, 12/5/2015, 3244 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1669

[Abstract] Several studies have shown that the detrimental influence of abdominal obesity on metabolic processes is mediated by the intra-abdominal fat depot. Visceral adipose tissue has been shown to be an independent risk factor for coronary heart disease, hypertension, impaired glucose tolerance and Diabetes Mellitus Type 2 (DM2). Diet-induced obesity in mice, primarily of the C57BL/6J strain, is a commonly used method to study the development of insulin resistance as a model for DM2. The white or visceral adipose tissue (here referred to as VAT), especially the fat around the gonads, is a commonly used organ of study in this model, as it accumulates large numbers of lymphocytes in response to diet-induced obesity. The protocol below describes the isolation of lymphocytes from the stromal vascular fraction (SVF) from VAT.

[Abstract]

Materials and Reagents

  1. 50 ml centrifuge tubes
  2. 70 µm cell strainer (BD Biosciences, Falcon®, catalog number: 352350 )
  3. Male mouse (e.g. C57BL/6J) 8-20 weeks old
    Note: Generally, male mice are more severely affected by type 2 diabetes than female mice, and they are used exclusively in diet-induced diabetes studies (www.jax.org).
  4. Collagenase from Clostridium histolyticum type IV (Sigma-Aldrich)
  5. Fetal Bovine Serum (FBS) (Pan biotech GmbH)
  6. Trypan blue
  7. RPMI 1640 (with L-glutamine; 25 mM Hepes; 2.2 g/L NaHCO3) (Pan Biotech GmbH)
  8. MilliQ water
  9. 0.83% NH4Cl
  10. 0.168% Na2CO3
  11. 1 mM EDTA (pH 7.3)
  12. 1x PBS (pH 7.3)
  13. 0.2% BSA
  14. 3% RPMI 1640 (see Recipes)
  15. 3% RPMI + 1 mg/ml Collagenase D (or IV) (see Recipes)
  16. Erylysis buffer (see Recipes)
  17. FACS wash buffer (see Recipes)

Equipment

  1. Soft wood tablet and pins
  2. Disinfectant
  3. Small thin surgical scissors and tweezers
  4. Thermostatic shaker
  5. Vortex
  6. Vacuum pump
  7. Centrifuge

Procedure

  1. Euthanize the mouse by O2/CO2 (70%/30%) intoxication, followed by CO2 asphyxiation. Of note, all experiments using mice were approved beforehand by your Institutional Animal Care and Use Committee and were in accordance with national and international guidelines.
  2. Gently lay down the mouse on its back, on a soft wood surface, stretch the limbs and fix the four paws with pin (Figure 1).


    Figure 1. Fixation. Fix the animal on its back to facilitate VAT removal.

  3. Clean the abdomen with disinfectant.
  4. Use a scissor to make a midline incision (Figure 2) and use straight tweezers to retract the skin. Open the muscular wall with another cutting tool. Steps 4-6 are also illustrated in Video 1.

    Video 1. Excision of VAT

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


    Figure 2. Opening of the skin.
    First remove the skin to prevent damage to the internal organ by cutting too deep.

  5. The peritoneum contains several adipose tissue reservoirs. Draw out the white or visceral (perigonadal) adipose tissue (VAT). The perigonadal fat expands most vigorously upon high-fat feeding (Figure 3).


    Figure 3. Identification of the VAT. The perigonadal VAT is located in the lower half of the abdomen, surrounding the gonads.

  6. Cut out the VAT carefully along the epididymis and vas deferens (in males) or along the uterus (in females). Take care not to excise any part of the gonads (Figure 4) (Supplementary Figure 1).


    Figure 4. Isolation of VAT. Gently cut out the VAT without damaging the gonads (indicated by arrow).

  7. Slice the VAT into small parts (of approximately 3 mm) with a scissors and put them in a 50 ml tube (Figure 5), containing 5 ml of freshly prepared 3% RPMI with 1 mg/ml Collagenase D (or IV).
    Note that most collagenases are sold as a mixture of proteins and are not a purified enzyme. Some collagenase batches may therefore contain other protease activity and can digest cell surface proteins. We recommend testing the effects of every collagenase batch on your proteins of interest. For example by comparing the expression of your proteins of interest on splenic lymphocytes by flow cytometry with and without collagenase treatment.


    Figure 5. Digestion of the VAT. Place the VAT in 50 ml tube containing 5 ml of freshly prepared 3% RPMI with 1 mg/ml Collagenase D for digestion after cutting it in small pieces using a scalpel or scissors.

  8. Incubate the tissue in a thermostatic shaker for 1 hour at 37 °C, shaking at 270 rpm (Figure 6).


    Figure 6. Incubation. Place the tubes for digestion in a heated, shaking incubator.

  9. Vortex the tube and add 5 ml of fresh, cold 3% RPMI.
  10. Centrifuge at 500 x g for 5 min. Remove floating adipocytes using a vacuum pump and afterwards carefully discard the remaining supernatant by inverting the tube (Figure 7).


    Figure 7. Removal of supernatant after digestion and centrifugation. The pellet contains leukocytes, stromal cells and remaining erythrocytes, which are collectively called the Stromal Vacular Fraction (SVF). The supernatant contains a liquid phase (with debris) and an oil phase and possibly a fat phase.

  11. In order to eliminate erythrocytes, resuspend the pellet in 1ml of hypotonic solution (Erylysis buffer), vortex and leave for 3 min at room temperature.
  12. Run the suspension over a 70 µm cell strainer placed on a 2 ml Eppendorf tube containing 500 µl of cold 3% RPMI (Figure 8).


    Figure 8. Removal of debris. The suspension is run over a sieve to get rid of debris from connective tissue and lysed erythrocytes.

  13. Vortex briefly and centrifuge at 500 x g for 5 min at room temperature.
  14. Remove the supernatant and resuspend the pellet in 250 µl 3% RPMI.
  15. Count viable cells using trypan blue dye exclusion. You can expect between 500,000 and 1,000,000 cells per fat pad.

Representative data


Figure 9. FACS plot of Leukocytes isolated from VAT. Cells were stained with viable dye [Propidium Iodide (PI)] and CD45 antibodies. Gated is for singlets.

Supplementary figures


Supplementary Figure 1a. Gonadal/visceral adipose tissue in female mice



Supplementary Figure 1b. Gonadal/visceral adipose tissue in male mice

Recipes

  1. 3% RPMI 1640
    For 100 ml of buffer, add 3 ml of heat-inactivated FBS to 97 ml RPMI 1640 and
    Refrigerate at 4 °C before use
  2. 3% RPMI 1640 + 10 μg/ml Collagenase IV
    For 100 ml of buffer, add 3 ml of heat-inactivated FBS and 1 ml of Collagenase IV (0.1 mg/ml) to 96 ml of RPMI 1640 and Refrigerate at 4 °C before use
  3. Erylysis buffer
    500 ml MilliQ water
    0.83% NH4Cl
    0.168% Na2CO3
    1 mM EDTA (pH 7.3)
    Sterile filtration
  4. FACS wash buffer (pH 7.0-8.0)
    1x PBS (pH 7.3)
    0.2% BSA
    1 mM EDTA

Acknowledgements

When using this protocol, please refer to Wensveen et al. (2015). This work is supported by the European Foundation for the Study of Diabetes (New Horizons Program), the Unity through Knowledge Fund (15/13 to B. P.), the University of Rijeka (13.06.1.1.03 to B. P.), the EU ESFEuropean Social Fund - ES (HR.3.2.01-0263 to B.P.), the Netherlands Organization for Scientific Research (91614029 to F. M. W.) and the European Commission (PCIG14-GA-2013-630827 to F. M. W.).

References

  1. Ng, A. C., Wai, D. C., Tai, E. S., Ng, K. M. and Chan, L. L. (2012). Visceral adipose tissue, but not waist circumference is a better measure of metabolic risk in Singaporean Chinese and Indian men. Nutr Diabetes 2: e38.
  2. Wajchenberg, B. L. (2000). Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 21(6): 697-738.
  3. Wensveen, F. M., Jelencic, V., Valentic, S., Sestan, M., Wensveen, T. T., Theurich, S., Glasner, A., Mendrila, D., Stimac, D., Wunderlich, F. T., Bruning, J. C., Mandelboim, O. and Polic, B. (2015). NK cells link obesity-induced adipose stress to inflammation and insulin resistance. Nat Immunol 16(4): 376-385.

材料和试剂

  1. 50ml离心管
  2. 70μm细胞滤器(BD Biosciences,Falcon ,目录号:352350)
  3. 8-20周龄的雄性小鼠( C57BL/6J)
    注意:通常,雄性小鼠比雌性小鼠更严重地受2型糖尿病的影响,并且它们仅用于饮食诱导的糖尿病研究(www.jax.org)。
  4. 来自溶组织梭菌IV型的胶原酶(Sigma-Aldrich)
  5. 胎牛血清(FBS)(Pan biotech GmbH)
  6. 台盼蓝
  7. RPMI 1640(具有L-谷氨酰胺; 25mM Hepes; 2.2g/L NaHCO 3)(Pan Biotech GmbH)
  8. MilliQ水
  9. 0.83%NH 4 Cl /
  10. 0.168%Na 2 CO 3 sub/
  11. 1mM EDTA(pH7.3)
  12. 1x PBS(pH 7.3)
  13. 0.2%BSA
  14. 3%RPMI 1640(见配方)
  15. 3%RPMI + 1mg/ml胶原酶D(或IV)(见配方)
  16. 溶解缓冲液(参见配方)
  17. FACS洗涤缓冲液(参见配方)

设备

  1. 软木片和钉
  2. 消毒剂
  3. 小型手术剪刀和镊子
  4. 恒温振动器
  5. 涡流
  6. 真空泵
  7. 离心机

程序

  1. 通过O 2/CO 2(70%/30%)中毒对小鼠进行安乐死,接着进行CO 2窒息。值得注意的是,所有使用小鼠的实验都由您的机构动物护理和使用委员会事先批准,并且符合国家和国际指南。
  2. 轻轻地将鼠标放在它的背部,在柔软的木材表面,伸展四肢,用钉固定四个爪子(图1)。


    图1.固定。将动物固定在背部,以方便增值税的移除。

  3. 用消毒剂清洁腹部。
  4. 使用剪刀做一个中线切口(图2),并使用直镊子缩回皮肤。用另一个切割工具打开肌肉墙。视频1中还显示了步骤4-6。

    视频1. 切除增值税
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    图2.皮肤开口。首先取下皮肤,以防止切割太深对内脏器官造成伤害。

  5. 腹膜含有几个脂肪组织储库。绘制白色或内脏(围产期)脂肪组织(VAT)。定期脂肪在高脂肪喂养时最强烈地膨胀(图3)。


    图3.增值税的识别。性腺增值税位于腹部下半部,围绕性腺。

  6. 沿附睾和输精管(男性)或沿子宫(女性)切除增值税。注意不要去除性腺的任何部分(图4)(补充图1)。


    图4.增值税的隔离。轻轻裁减增值税,而不损坏性腺(箭头所示)。

  7. 用剪刀将增值税切成小部分(约3毫米),并将他们放入50ml管(图5),包含5ml新鲜制备的3%RPMI与1毫克/毫升胶原酶D(或IV)。
    请注意,大多数胶原酶是作为蛋白质的混合物出售的,不是纯化的酶。因此,一些胶原酶批次可以含有其他蛋白酶活性并且可以消化细胞表面蛋白。我们建议测试每个胶原酶批次对您感兴趣的蛋白质的影响。例如通过用和不用胶原酶处理的流式细胞术比较感兴趣的蛋白质在脾淋巴细胞上的表达。


    图5.增值税的消化。将增值税放入50ml管中,该管含有5ml新鲜制备的3%RPMI和1mg/ml胶原酶D用于消化,使用手术刀切割成小块剪刀。

  8. 在恒温振荡器中在37℃下孵育组织1小时,以270rpm振摇(图6)。


    图6.孵育。将消化管置于加热的摇动培养箱中。

  9. 涡旋管,加入5毫升新鲜,冷3%RPMI
  10. 在500×g离心5分钟。使用真空泵除去浮动脂肪细胞,然后通过倒置管仔细丢弃剩余的上清液(图7)

    图7.消化和离心后上清液的去除。沉淀含有白细胞,基质细胞和剩余的红细胞,统称为基质空泡分数(SVF)。上清液含有液相(具有碎片)和油相以及可能的脂肪相。

  11. 为了消除红细胞,将沉淀物重悬在1ml低渗溶液(Erylysis缓冲液)中,涡旋并在室温下放置3分钟。
  12. 将悬浮液在置于含有500μl冷的3%RPMI的2ml Eppendorf管上的70μm细胞过滤器上运行(图8)。


    图8.去除碎片。将悬浮液过筛,以除去结缔组织和裂解红细胞的碎片。

  13. 短暂涡旋并在室温下以500×g离心5分钟
  14. 取出上清液,并将沉淀重悬在250μl3%RPMI中。
  15. 使用台盼蓝染料排除计数活细胞。你可以期望每个脂肪垫在500,000和1,000,000个细胞之间。

代表数据


图9.从增值税中分离的白细胞的FACS图。 用活的染料[碘化丙啶(PI)]和CD45抗体染色细胞。门是为单片。

补充数字


补充图1a。雌性小鼠的性腺/内脏脂肪组织



补充图1b。雄性小鼠性腺/内脏脂肪组织

食谱

  1. 3%RPMI 1640
    对于100ml缓冲液,将3ml热灭活的FBS加入到97ml RPMI 1640和
    中 使用前在4°C冷藏
  2. 3%RPMI 1640 +10μg/ml胶原酶IV
    对于100ml缓冲液,在使用前在4℃下向96ml RPMI 1640和Refrigerate中加入3ml热灭活的FBS和1ml胶原酶IV(0.1mg/ml)
  3. 溶解缓冲液
    500 ml MilliQ水
    0.83%NH 4 Cl / 0.168%Na 2 CO 3 sub/
    1mM EDTA(pH7.3) 无菌过滤
  4. FACS洗涤缓冲液(pH 7.0-8.0)
    1x PBS(pH 7.3)
    0.2%BSA 1mM EDTA

致谢

使用此协议时,请参阅Wensveen等人(2015)。这项工作是由欧洲糖尿病研究基金会(新视野计划),通过知识基金(15/13到BP),里耶卡大学(13.06.1.1.03到BP),欧盟ESFEuropean社会基金 - ES(HR.3.2.01-0263至BP),荷兰科学研究组织(91614029至FMW)和欧洲委员会(PCIG14-GA-2013-630827至FMW)。

参考文献

  1. Ng,A.C.,Wai,D.C.,Tai,E.S.,Ng,K.M.and Chan,L.L。(2012)。 内脏脂肪组织,但不是腰围是新加坡中国和印度男性代谢风险的更好衡量标准。 Nutr Diabetes 2:e38。
  2. Wajchenberg,B.L。(2000)。 皮下和内脏脂肪组织:它们与代谢综合征的关系 Endocr Rev 21(6):697-738
  3. Wensveen,FM,Jelencic,V.,Valentic,S.,Sestan,M.,Wensveen,TT,Theurich,S.,Glasner,A.,Mendrila,D.,Stimac,D.,Wunderlich,FT,Bruning,JC ,Mandelboim,O.和Polic,B。(2015)。 NK细胞将肥胖诱导的脂肪应激与炎症和胰岛素抵抗联系起来。 Nat Immunol 16(4):376-385
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How to cite this protocol: Valentić, S., Wensveen, F. M. and Polić, B. (2015). Isolation of Lymphocytes from Murine Visceral Adipose Tissue. Bio-protocol 5(23): e1669. DOI: 10.21769/BioProtoc.1669; Full Text



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