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Protocol for Murine/Mouse Platelets Isolation and Their Reintroduction in vivo

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Mar 2012



Platelets and coagulation have long been known to be essential for metastasis in experimental models. In order to study the interactions between tumor cells, platelets and endothelium, we have adapted methods used in coagulation research for the isolation of platelets and their reintroduction into mice. Anti-coagulated murine blood served as the source for platelets. Platelets were separated from other elements of the whole blood by centrifugation. Here the critical elements are first inhibition of coagulation and second isolation and maintenance of the platelets in the presence of inhibitors of platelet activation. We then used the vital dye PKH26 to fluorescently label the platelets. Infusion of these labelled platelets allows microscopic observation of the introduced platelets. After reintroduction, these platelets appear to function normally and comprise approximately 50% of the total platelets. Because they are fluorescently labelled, they can easily be identified. Finally it would be possible to use these methods for the determination of specific effects of altered gene expression in platelets by using platelets from genetically engineered mice. These methods have facilitated study of the interactions between platelets and tumor cells in tissue culture and in murine models. They would also be applicable to video microscopy. Here we provide details of the methods we have used for platelet isolation from mice and their staining for further microscopy and re-introduction into mice.

Keywords: Platelets (血小板), Coagulation (凝血), Fluorescent labeling (荧光标记), Vital dye (活体染料), Clot (凝结)


Platelets are known to be essential for metastasis, but also to play roles during tumor growth not to mention clot formation. In order to readily identify and track platelets we developed the means for fluorescently labeling and reinfusing platelets. This allows them to be readily identified in tissues without immunostaining. Using these methods, we showed that interactions between tumor cells and platelets play key roles in survival of the tumour cells early during metastasis (Im et al., 2004; Gil-Bernabe et al., 2012). Platelets formed clots with tumor cells in the blood stream and this coagulation promoted spreading and subsequent retention of the tumor cells during lung metastasis (Im et al., 2004). Tissue factor expressed by tumor cells is capable of mediating clot formation with platelets and recruitment of macrophages (Gil-Bernabe et al., 2012). The interaction of platelets, thrombin and fibrin also have been reported to promote metastasis by generating epithelial-mesenchymal transition of the cancer cells and evasion from the immune system by protection from NK cells as well as secretion of pro-metastatic chemokines and cytokines (Labelle et al., 2011; Nieswandt et al., 1999; Palumbo et al., 2005 and 2007). Precise tracking of platelets will provide opportunities to uncover how tumor cells utilize the host for their survival.

Materials and Reagents

  1. Scalpel (Swann Morton, catalog number: 0208 )
  2. Syringe, 1 ml (BD, catalog number: 300013 )
  3. 15 ml conical bottom polypropylene tube (SARSTEDT, catalog number: 62.554.502 )
  4. 5 ml pipette
  5. Needle (27 G)
  6. Mice (4-6 weeks old, weight over 20 g, Charles Liver, UK)
  7. Isoflurane (Abbott, catalog number: 0044-5260-05 )
  8. EGTA (Sigma-Aldrich, catalog number: E3889 )
  9. PKH26 Kit (Sigma-Aldrich, catalog number: PKH26GL-1KT )
  10. Sodium chloride, NaCl (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10378573 )
  11. Potassium chloride, KCl (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10427460 )
  12. Sodium phosphate monobasic monohydrate, NaH2PO4·H2O (Sigma-Aldrich, catalog number: S9638 )
  13. HEPES (Sigma-Aldrich, catalog number: H3375 )
  14. Glucose (Thermo Fisher Scientific, GibcoTM, catalog number: 15023021 )
  15. Magnesium chloride, MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  16. Trisodium citrate (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10362234 )
  17. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  18. Dextrose (Thermo Fisher Scientific, Fisher Scientific, catalog number: D16-1 )
  19. Prostaglandin E1 (Sigma-Aldrich, catalog number: P5515 )
  20. 100% ethanol
  21. Sodium bicarbonate, NaHCO3 (Thermo Fisher Scientific, Fisher Scientific, catalog number: S637-212 )
  22. Distilled water
  23. Bovine serum albumin, BSA (Sigma-Aldrich, catalog number: A2058 )
  24. Sodium bicarbonate, Na2HPO4 (Thermo Fisher Scientific, Fisher Scientific, catalog number: S374 )
  25. Sodium citrate (Sigma-Aldrich, catalog number: 71498 )
  26. Modified Tyrode’s calcium-free buffer (see Recipes)
  27. ACD buffer (see Recipes)
  28. Undiluted prostaglandin E1 solution
  29. Resuspension buffer (see Recipes)
  30. Washing buffer (see Recipes)
  31. Citrate-albumin buffer (see Recipes)


  1. Coulter counter (CDC Technology, model: HEMAVET® 1500 )
  2. Centrifuge (Thermo Fisher Scientific, model: Jouan CR4i )


  1. Platelet isolation
    1. The mouse is placed under terminal anaesthesia by inhalation of isoflurane (2.5% with oxygen at medical grade).
    2. With a scalpel the abdomen is opened and the bowel pushed aside to expose the major abdominal vessels.
    3. A syringe with a needle (27 G) containing ACD buffer (150 µl/ml blood) is inserted into the vena cava above the renal veins to collect blood (Figure 1). Blood volume collected in this way is usually from 800-1,000 µl for a mouse of 20.0 g weight. After the collection of blood, the mouse was completely exsanguinated by transection of the vena cava.

      Figure 1. Blood collection from the vena cava in a mouse. A shows position of the vena  cava and B shows blood collection with a syringe containing ACD buffer.

    4. The blood should be anti-coagulated by rapid mixing with the ACD buffer during collection. The blood is then transferred to a 15 ml conical bottom polypropylene tube and mixed with washing buffer (3 ml/ml, 1 ml of 100 mmol/L EGTA [pH 6.8] and 2 ml of resuspension buffer).
    5. The tube is centrifuged at 180 x g at 22 °C for 10 min. This results in pelleting of the larger cellular elements such as red blood cells and leukocytes.
    6. Platelet-rich plasma is collected and transferred to a 15 ml conical bottom polypropylene tube. The pellet discarded. The washing buffer detailed in step A4 now with the addition of prostaglandin E1 (0.25 µmol/L) is added to yield a total volume of 10 ml. The mixture was gently pipetted 3-5 times with a 5 ml pipette. The prostaglandin is essential at this point to prevent platelet activation.
    7. The tube is centrifuged at 1,250 x g and 22 °C for 10 min.
    8. The pellet now consists mainly of platelets. Remove the supernatant and wash two more times using the buffer in step A6 with prostaglandin and centrifuge as in step A7. 
    9. After suspension at this point in 10 ml of washing buffer including 0.25 µmol/L prostaglandin E1 the platelets can be counted.  
    10. To count the platelets, mix the platelet suspension (15 µl) and undiluted prostaglandin E1 (5 µl, 500 µM). Counting can be done in a Coulter counter. Note that the counter must be adjusted for the size of mouse platelets. The diameter of the mouse platelet is approximately 0.5 µm, which is smaller than human platelets (1-2 µm).
    11. At this point the platelets can be used for experimentation remembering that until an experiment is initiated, it is important to block activation with 0.25 µmol/L prostaglandin E1. This will then need to be removed- often by dilution- to assess platelet action.

  2. Platelet staining
    1. We have used the vital dye PKH26 to fluorescently tag the collected platelets following the manufacturer’s directions with some modifications. At this point the platelets from step A9 can be centrifuged as in A7. They should be resuspended in 1 ml of Diluent C (PKH26 Kit) to be 8 x 105/µl with added 0.25 µmol/L prostaglandin E1.
    2. Prepare a stock solution of PKH26 (1 mM) by dilution of PKH26 dye solution in 100% ethanol.
    3. Add 2 µl of both PKH26 and prostaglandin E1 (2.5 µg/ml) into 1 ml of platelet suspension.
    4. Immediately mix the sample by gentle pipetting.
    5. Incubate the sample at 22 °C for 10 min.
    6. The staining reaction is stopped by addition of 10 ml of a mixture of citrate-albumin buffer, 0.35% bovine serum albumin (pH 6.5) and 0.25 µmol/L prostaglandin E1.
    7. Incubate the mixture at 22 °C for 1 min.
    8. Pellet the platelets by centrifugation at 1,250 x g at 22 °C for 20 min.
    9. Remove the supernatant and resuspend the platelets at 6 x 106/µl in the resuspension buffer for use (as described in Recipe 4).
    10. For in vivo experiments, 100 µl of platelet suspended solution can be injected with a 1 ml syringe a needle (27 G) into the mouse tail vein.
    11. We find that the number of platelets harvested in this way from 5 mice usually supplies enough for in vivo injections into 3 mice. The resultant reconstitution usually results in approximately 50% of the platelets being newly introduced and fluorescent. They remain functional for at least 24 h. We have not looked in detail after that time, but in principle a longer time span should be possible.

Data analysis

The details of data analysis are presented at the article by Im et al. (2004) and Gil-Bernabe et al. (2012).


PGE1 must be added freshly for each use of the buffer and cannot be stockpiled. The most variable part of the procedure is the reinjection of platelets intravenously.


  1. Modified Tyrode’s calcium-free buffer (pH 7.2)
    134 mmol/L NaCl
    3 mmol/L KCl
    0.3 mmol/L NaH2PO4·H2O
    5 mmol/L HEPES
    5 mmol/L glucose
    2 mmol/L MgCl2
    Adjust pH to 7.2 with HCl (36.5%-38%)
  2. ACD buffer (150 µl/ml blood, pH 4.5)
    85 mmol/L trisodium citrate
    71 mmol/L citric acid
    111 mmol/L dextrose
    Note: The solution has a pH of about 4.5 and thus adjustment does not needed.
  3. Undiluted prostaglandin E1 solution
    50 µmol prostaglandin E1/100 ml ethanol
  4. Resuspension buffer
    100 ml of 7.5% NaHCO3
    133 ml of distilled water
    300 mg of bovine serum albumin
    15 ml modified Tyrode’s calcium-free buffer
  5. Washing buffer
    1 ml of 100 mmol/L EGTA (pH 6.8)
    2 ml of resuspension buffer
  6. Citrate-albumin buffer (pH 6.5)
    11 mmol/L glucose
    128 mmol/L NaCl
    4.3 mmol/L NaH2PO4·H2O
    7.5 mmol/L Na2HPO4
    4.8 mmol/L sodium citrate
    2.4 mmol/L citric acid
    0.35 % (w/v) bovine serum albumin
    Adjust pH to 6.5 with HCl (36.5%-38%)


The authors were supported by NIH grants R01 CA89188, R01 CA46830 and currently are funded by Cancer Research UK. This protocol was adapted from the article by Im et al. (2004).


  1. Gil-Bernabe, A. M., Ferjancic, S., Tlalka, M., Zhao, L., Allen, P. D., Im, J. H., Watson, K., Hill, S. A., Amirkhosravi, A., Francis, J. L., Pollard, J. W., Ruf, W. and Muschel, R. J. (2012). Recruitment of monocytes/macrophages by tissue factor-mediated coagulation is essential for metastatic cell survival and premetastatic niche establishment in mice. Blood 119(13): 3164-3175.
  2. Im, J. H., Fu, W., Wang, H., Bhatia, S. K., Hammer, D. A., Kowalska, M. A. and Muschel, R. J. (2004). Coagulation facilitates tumor cell spreading in the pulmonary vasculature during early metastatic colony formation. Cancer Res 64(23): 8613-8619.
  3. Labelle, M., Begum, S. and Hynes, R. O. (2011). Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell 20(5): 576-590.
  4. Nieswandt, B., Hafner, M., Echtenacher, B. and Mannel, D. N. (1999). Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res 59(6): 1295-1300.
  5. Palumbo, J. S., Talmage, K. E., Massari, J. V., La Jeunesse, C. M., Flick, M. J., Kombrinck, K. W., Jirouskova, M. and Degen, J. L. (2005). Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 105(1): 178-185.
  6. Palumbo, J. S., Talmage, K. E., Massari, J. V., La Jeunesse, C. M., Flick, M. J., Kombrinck, K. W., Hu, Z., Barney, K. A. and Degen, J. L. (2007). Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and-independent mechanisms. Blood 110(1): 133-141.



背景 已知血小板对于转移是必需的,而且在肿瘤生长期间也起作用,更不用说凝块形成。为了容易识别和追踪血小板,我们开发了荧光标记和再灌注血小板的手段。这使得它们可以容易地在不需要免疫染色的组织中鉴定。使用这些方法,我们显示肿瘤细胞和血小板之间的相互作用在转移早期在肿瘤细胞的存活中起关键作用(Im等人,2004; Gil-Bernabe等人,2012)。血小板在血液中形成具有肿瘤细胞的凝块,并且这种凝血促进肿瘤细胞在肺转移期间的扩散和随后的保留(Im等人,2004)。由肿瘤细胞表达的组织因子能够介导血小板形成和巨噬细胞的募集(Gil-Bernabe等人,2012)。据报道血小板,凝血酶和纤维蛋白的相互作用通过从NK细胞的保护以及前转移趋化因子和细胞因子的分泌产生癌细胞的上皮 - 间质转化和逃避免疫系统来促进转移(Labelle ,2011; Nieswandt等人,1999; Palumbo等人,2005和2007)。血小板的精确跟踪将提供发现肿瘤细胞如何利用宿主生存的机会。

关键字:血小板, 凝血, 荧光标记, 活体染料, 凝结


  1. Scalpel(Swann Morton,目录号:0208)
  2. 注射器,1 ml(BD,目录号:300013)
  3. 15毫升锥形底部聚丙烯管(SARSTEDT,目录号:62.554.502)
  4. 5ml移液器
  5. 针(27 G)
  6. 小鼠(4-6周龄,体重超过20g,英国Charles Liver)
  7. 异氟烷(Abott,目录号:0044-5260-05)
  8. EGTA(Sigma-Aldrich,目录号:E3889)
  9. PKH26试剂盒(Sigma-Aldrich,目录号:PKH26GL-1KT)
  10. 氯化钠,NaCl(Thermo Fisher Scientific,Fisher Scientific,目录号:10378573)
  11. 氯化钾,KCl(Thermo Fisher Scientific,Fisher Scientific,目录号:10427460)
  12. 磷酸二氢钠一水合物,NaH 2 PO 4 H 2 O(Sigma-Aldrich,目录号:S9638)
  13. HEPES(Sigma-Aldrich,目录号:H3375)
  14. 葡萄糖(Thermo Fisher Scientific,Gibco TM ,目录号:15023021)
  15. 氯化镁,MgCl 2(Sigma-Aldrich,目录号:M8266)
  16. 柠檬酸三钠(Thermo Fisher Scientific,Fisher Scientific,目录号:10362234)
  17. 柠檬酸(Sigma-Aldrich,目录号:251275)
  18. 右旋糖(Thermo Fisher Scientific,Fisher Scientific,目录号:D16-1)
  19. 前列腺素E1(Sigma-Aldrich,目录号:P5515)
  20. 100%乙醇
  21. 碳酸氢钠,NaHCO 3(Thermo Fisher Scientific,Fisher Scientific,目录号:S637-212)
  22. 蒸馏水
  23. 牛血清白蛋白,BSA(Sigma-Aldrich,目录号:A2058)
  24. 碳酸氢钠,Na 2 HPO 4(Thermo Fisher Scientific,Fisher Scientific,目录号:S374)
  25. 柠檬酸钠(Sigma-Aldrich,目录号:71498)
  26. 修改Tyrode的无钙缓冲液(见配方)
  27. ACD缓冲(见配方)
  28. 未稀释的前列腺素E1溶液
  29. 再悬浮缓冲液(见配方)
  30. 洗涤缓冲液(见配方)
  31. 柠檬酸 - 白蛋白缓冲液(参见食谱)


  1. 库尔特计数器(CDC Technology,型号:HEMAVET ® 1500)
  2. 离心机(Thermo Fisher Scientific,型号:Jouan CR4i)


  1. 血小板分离
    1. 通过吸入异氟烷(2.5%,医用级别的氧气)将小鼠置于终端麻醉下。
    2. 用手术刀打开腹部,将肠子推开,露出主要的腹部血管。
    3. 将含有ACD缓冲液(150μl/ml血液)的针头(27G)的注射器插入肾静脉上方的腔静脉以收集血液(图1)。以这种方式收集的血容量对于20.0g重量的小鼠通常为800-1,000μl。收集血液后,小鼠通过切断腔静脉完全放血。

      图1.小鼠中腔静脉的血液收集。 A显示腔静脉的位置,B显示采用含有ACD缓冲液的注射器采集血液。

    4. 血液应在收集过程中通过与ACD缓冲液快速混合来抗凝。然后将血液转移到15ml锥形底部聚丙烯管中,并与洗涤缓冲液(3ml/ml,1ml 100mmol/L EGTA [pH6.8]和2ml再悬浮缓冲液)混合。
    5. 将管在180℃下以22℃离心10分钟。这导致较大的细胞元素如红细胞和白细胞的沉淀
    6. 收集富含血小板的血浆并转移到15ml锥形底部聚丙烯管中。弃粒。加入步骤A4中详述的洗涤缓冲液,加入前列腺素E1(0.25μmol/L),得到总体积为10ml。将混合物用5ml移液管轻轻移液3-5次。在这一点上,前列腺素是必不可少的,以防止血小板活化。
    7. 将管以1,250×g×22℃离心10分钟
    8. 丸粒现在主要由血小板组成。取出上清液,再用步骤A6中的缓冲液用前列腺素洗涤两次,如步骤A7所示离心。
    9. 此后在10ml洗涤缓冲液(包括0.25μmol/L前列腺素E1)中悬浮后,可以计数血小板。
    10. 计数血小板,混合血小板悬浮液(15μl)和未稀释的前列腺素E1(5μl,500μM)。可以在库尔特柜台进行计数。请注意,必须根据小鼠血小板的大小调整计数器。小鼠血小板的直径约为0.5μm,小于人血小板(1-2μm)。
    11. 此时血小板可用于实验记忆,直到开始实验,重要的是用0.25μmol/L前列腺素E1阻断活化。这样就需要通过稀释来去除血小板作用。

  2. 血小板染色
    1. 我们使用重要的染料PKH26按照制造商的指示对收集的血小板进行荧光标记,并进行了一些修改。在这一点上,来自步骤A9的血小板可以如A7那样离心。应将其悬浮于1ml稀释剂C(PKH26试剂盒)中,加入加入0.25μmol/L前列腺素E1的8×10 5 /μl/μl。
    2. 通过将PKH26染料溶液稀释在100%乙醇中制备PKH26(1mM)的储备溶液
    3. 将2μlPKH26和前列腺素E1(2.5μg/ml)加入1ml血小板悬浮液中。
    4. 通过轻轻移液器立即混合样品。
    5. 在22℃下孵育样品10分钟
    6. 通过加入10ml柠檬酸盐 - 白蛋白缓冲液,0.35%牛血清白蛋白(pH6.5)和0.25μmol/L前列腺素E1的混合物来终止染色反应。
    7. 将混合物在22℃下孵育1分钟。
    8. 通过在22℃下以1,250×g离心20分钟来造粒血小板。
    9. 取出上清液,并将重悬浮缓冲液中的6×10 6 /μl的血小板重新悬浮使用(如方案4所述)。
    10. 对于体内实验,可以将100μl血小板悬浮液注入1ml注射器针(27G)到小鼠尾静脉中。
    11. 我们发现,从5只小鼠以这种方式收获的血小板数目通常足以在3只小鼠体内注射。所得的重组通常导致约50%的血小板被新引入和荧光。它们保持功能至少24小时。在那段时间之后,我们还没有详细说明,但是原则上应该有更长的时间跨度。


数据分析的细节在Im等人的文章中给出。 (2004)和Gil-Bernabe等人。 (2012)。




  1. 改良的Tyrode的无钙缓冲液(pH 7.2)
    134 mmol/L NaCl
    3 mmol/L KCl
    0.3mmol/L NaH 2 PO 4 H 2 O 2//O 2 5 mmol/L HEPES
    5 mmol/L葡萄糖
    2mmol/L MgCl 2
  2. ACD缓冲液(150μl/ml血液,pH 4.5) 85 mmol/L柠檬酸三钠
    71 mmol/L柠檬酸 111 mmol/L葡萄糖
  3. 未稀释的前列腺素E1溶液
  4. 再悬浮缓冲液
    100ml 7.5%NaHCO 3
  5. 洗涤缓冲液
    1ml 100mmol/L EGTA(pH6.8)
    2 ml再悬浮缓冲液
  6. 柠檬酸 - 白蛋白缓冲液(pH6.5)
    11 mmol/L葡萄糖 128 mmol/L NaCl
    4.3 mmol/L NaH 2 PO 4 H 2 O 2// 7.5 mmol/L Na 2 HPO 4
    4.8 mmol/L柠檬酸钠 2.4 mmol/L柠檬酸
    0.35%(w/v)牛血清白蛋白 用HCl调节pH至6.5(36.5%-38%)


作者得到NIH授权R01 CA89188,R01 CA46830的支持,目前由英国癌症研究所资助。该协议是由Im等人的文章改编而成。 (2004)。


  1. Gil-Bernabe,AM,Ferjancic,S.,Tlalka,M.,Zhao,L.,Allen,PD,Im,JH,Watson,K.,Hill,SA,Amirkhosravi,A.,Francis,JL,Pollard,JW ,Ruf,W.和Muschel,RJ(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22327225"target ="_ blank" >通过组织因子介导的凝血对单核细胞/巨噬细胞的募集对小鼠的转移性细胞存活和转移前的小生境建立至关重要。血液 119(13):3164-3175。
  2. Im,JH,Fu,W.,Wang,H.,Bhatia,SK,Hammer,DA,Kowalska,MA和Muschel,RJ(2004)。凝血促进早期转移性集落形成期间肺血管中的肿瘤细胞扩散。癌症研究 64 (23):8613-8619。
  3. Labelle,M.,Begum,S。和Hynes,RO(2011)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22094253"target ="_ blank">血小板和癌细胞之间的直接信号传导引起上皮 - 间质样转化并促进转移。 20(5):576-590。
  4. Nieswandt,B.,Hafner,M.,Echtenacher,B.and Mannel,DN(1999)。  小鼠中天然杀伤细胞的肿瘤细胞裂解受血小板阻碍。 59(6):1295-1300。
  5. Palumbo,JS,Talmage,KE,Massari,JV,La Jeunesse,CM,Flick,MJ,Kombrinck,KW,Jirouskova,M.and Degen,JL(2005)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/15367435"target ="_ blank">通过阻止自然杀伤细胞介导的肿瘤细胞消除,血小板和纤维蛋白(ogen)增加转移潜能。血液 105(1):178-185。
  6. Palumbo,JS,Talmage,KE,Massari,JV,La Jeunesse,CM,Flick,MJ,Kombrinck,KW,Hu,Z.,Barney,KA和Degen,JL(2007)。< a class =插入文件"href ="http://www.ncbi.nlm.nih.gov/pubmed/17371949"target ="_ blank">肿瘤细胞相关组织因子和循环止血因子通过自然杀伤细胞依赖性协同增加转移潜力独立的机制。 110(1):133-141。
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引用:Im, J. H. and Muschel, R. J. (2017). Protocol for Murine/Mouse Platelets Isolation and Their Reintroduction in vivo. Bio-protocol 7(4): e2132. DOI: 10.21769/BioProtoc.2132.