Isolation of Lymphocytes from Murine Visceral Adipose Tissue

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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.

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)


  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


  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 pins (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

    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 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 into small pieces using a scalpel or scissors.

  8. Incubate the tissue in a thermostatic shaker for 1 h 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 Vascular 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


  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


When using this protocol, please refer to Wensveen et al. (2015). This work was 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 ( 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.).


  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.


几个研究已经表明,腹部肥胖对代谢过程的有害影响是由腹内脂肪库介导的。 内脏脂肪组织已被证明是冠心病,高血压,葡萄糖耐量降低和2型糖尿病(DM2)的独立危险因素。 小鼠,主要是C57BL/6J菌株的饮食诱导的肥胖是一种常用的方法来研究胰岛素抵抗的发展作为DM2的模型。 白色或内脏脂肪组织(这里称为VAT),特别是性腺周围的脂肪,是该模型中常用的研究的器官,因为它响应于饮食诱导的肥胖积累大量淋巴细胞。 以下方案描述了淋巴细胞从基质血管部分(SVF)中分离出VAT。


  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)
  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)


  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


  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)。


  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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  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)。


  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)。


  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)。


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


  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)


  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)。


  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。


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。





  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


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(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)。


  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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引用: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.

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