Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate

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Scientific Reports
Jul 2016



Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent reagent. O-phthalaldehyde has an ability to react specifically with GSH at pH 8 and GSSG at pH 12 respectively. N-ethylmaleimide (NEM) has been used to prevent auto-oxidation of GSH during measurement of GSSG in the present protocol. The original protocol by Hissin and Hilf was developed for glutathione estimation in Rat liver tissue. The present protocol has been standardized following Hissin and Hilf (1976) for the estimation of glutathione in cultured microglial cell lysate but it can also be used for other mammalian cell lysate. In our lab same protocol has been used for the estimation of glutathione in the whole cell lysate of murine neuroblastoma cell, N2a.

Keywords: Glutathione (谷胱甘肽), Antioxidant (抗氧化剂), Free radical (自由基), Microglia (N9) (小胶质细胞(N9)), o-Phthalaldehyde (邻苯二甲醛), N-ethylmaleimide (N—乙基马来酰亚胺)


This method was published by Hissin and Hilf in analytical biochemistry way back in 1976 (Hissin and Hilf, 1976). There are methods available to detect GSH accurately however; due to readily oxidative conversion of GSH into GSSG most of the methods give an overestimate of GSSG. Cohen and Lyle (1966) solved the problem by using NEM to prevent oxidative conversion of GSH into GSSG and also preventing GSH to react with OPT during GSSG estimation (Figures 1 and 2). We have used this simple and reliable method to detect GSH and GSSG in our experimental system (microglial cell lysate). The main advantage of this protocol is that, it does not involve sophisticated instrument like high performance liquid chromatography (HPLC) which also needs sufficient expertise to handle as compared to plate reader which is more commonly available and easy to operate (Rahman et al., 2006).

Figure 1. Schematic of chemical reaction during GSH estimation in the N9 cell lysate

Figure 2. Schematic of chemical reaction during GSSG estimation in the N9 cell lysate

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-1005-WB-C-L )
  2. 0.22 μm filter
  3. 6 well culture plate (SRL LIFE SCIENCES, catalog number: 30006 )
  4. Culture flask
  5. 1.5 ml centrifuge tube (Corning, Axygen®, catalog number: MCT-150-R )
  6. 0.5 ml tube (Corning, Axygen®, catalog number: 14-222-292 )
  7. 96 well plate (SPL life sciences, catalog number: 30096 )
  8. Disposable plastic cell scraper (SRL LIFE SCIENCES, catalog number: 90020 )
  9. Cell line: In the present protocol mouse microglial cell line, N9 has been used, which was kindly gifted by Dr. Anirban Basu, National Brain Research Centre (NBRC), India (Singh et al., 2016). N9 cell line was developed by retroviral transfection of primary microglia cells with the v-myc or v-mil oncogenes of the avian retrovirus MH2. Cells were cultured in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin-streptomycin in a 5% CO2 incubator at 37 °C
  10. DMEM/F12 (Sigma-Aldrich, catalog number: 56498C )
  11. Sodium bicarbonate
  12. Double distilled water
  13. Fetal bovine serum (FBS) (Genetix Biotech, Cell cloneTM, catalog number: CCS-500-SA-U )
  14. Penicillin-streptomycin (Pen-Strep) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
  15. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  16. Bradford reagent (Bio-Rad Laboratories, catalog number: 5000006 )
  17. Tri-chloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
  18. o-Phthalaldehyde (Sigma-Aldrich, catalog number: P1378 )
  19. N-ethylmaleimide (Sigma-Aldrich, catalog number: E3876 )
  20. Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251 )
  21. Glutathione oxidized (GSSG) (Sigma-Aldrich, catalog number: G4376 )
  22. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  23. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  24. Dipotassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  25. Potassium phosphate dibasic trihydrate (K2HPO4·3H2O)
  26. EDTA disodium salt (Sigma-Aldrich, catalog number: E5513 )
    Note: This product has been discontinued.
  27. 100% ethanol (Merck, catalog number: 1009831011 )
  28. Methanol (SRL Laboratories, catalog number. 65524 )
  29. 0.1 M potassium phosphate EDTA buffer (KPE buffer) (see Recipes)
  30. 50% trichloroacetic acid (see Recipes)
  31. o-Phthaldehyde solution (10 mg/ml) (see Recipes)
  32. 0.4 M N-ethylmaleimide (see Recipes)
  33. 0.1 N sodium hydroxide (see Recipes)


  1. 1 ml and 200 µl pipettes (Eppendorf)
  2. Table top centrifuge (Sigma-zentrifuges, model: Sigma 3-18KS )
  3. Microplate reader (BMG LABTECH, model: FLUOstar Omega )
  4. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Steri-CycleTM CO2 Incubators )
  5. Sonicator (Sonics & Materials, model: VC 505 )


  1. Mars data analysis software, ver. 1.01 (Data analysis software for Microplate reader)


Flow diagram of the procedure (Figure 3)

Figure 3. Flow diagram of the procedure for the estimation of glutathione in mouse microglial cell line, N9

  1. Media preparation and cell culture
    1. For 1 L medium, 12 g DMEM/F12, 2.44 g sodium bicarbonate is dissolved in 890 ml of double distilled water and then 100 ml FBS along with 10 ml Pen-Strep is added. Finally media is filtered through 0.22 μm filter.
    2. N9 cells are seeded at a density of 1 x 106/2 ml in a 6-well culture plate and grown for 16-18 h in complete culture medium (DMEM/F12 medium supplemented with 10% FBS and 1% Pen-Strep).
    3. N9 cells are passaged after every 48 h and used till 6-7 passage after thawing.
    Note: If any treatment is given, please ensure that dose and duration of treatment is not toxic for cells. 
  2. Cell harvesting and lysis
    1. Remove culture medium and wash the cells with phosphate buffered saline (PBS). Add ice cold 0.1 M potassium phosphate buffer with EDTA (KPE buffer, 1 ml) to the each well and cells are dislodged from the surface of culture flask with the help of cell scraper and collected in a 1.5 ml centrifuge tube.
    2. Centrifuge the cell suspension at 350 x g for 5 min at 4 °C.
    3. Resulting cell pellet is resuspended in 200 µl KPE buffer with 1% protease inhibitor cocktail (PIC) and lysed by sonication for 5 sec pulse at 25 W two times on ice.
    Note: Cell scrapper is preferred instead of enzymatic dissociation to minimize the handling time. Besides, for enzymatic dissociation cells needs incubation in 37 °C whereas ice cold buffer can be used while using scrapper which is desirable during preparing cell lysate to keep structure and function of the protein unaltered.
  3. Protein sample preparation and estimation
    After sonication, lysed samples are centrifuged at 18,000 x g for 10 min at 4 °C and the resulting supernatants are collected in separate pre-cooled 1.5 ml centrifuge tubes.
    Keep 10 µl samples in 0.5 ml tubes for protein estimation by Bradford reagent.
  4. Protein precipitation
    After protein estimation, 10 µg protein sample is precipitated. Initially 80 µl protein sample is mixed with 20 µl trichloroacetic acid (TCA) (50% stock concentration), vortexed and kept in ice for 10 min. Protein sample with TCA is centrifuged at 9,100 x g for 10 min at 4 °C, the supernatant is transferred into a fresh 1.5 ml centrifuge tube.
  5. GSH and GSSG estimation
    1. GSH estimation
      Mix 10 µl supernatant with equal volume of OPT (1 mg/ml) and 180 µl KPE buffer (pH-8) in a black 96 well plate.
    2. GSSG estimation
      1. Transfer 50 µl of the supernatant into a new centrifuge tube, add 0.5 µl N-ethylmaleimide (stock concentration: 4 M) and mix thoroughly.
      2. Incubate for 30 min at room temperature to inhibit GSH.
      3. Add 10 µl of this sample, 10 µl OPT and 180 µl 0.1 N NaOH (pH-12) in a black 96 well plate.
        Note: Use multichannel pipette to add OPT and KPE buffer mixture in the 96 well plate to minimize the time lapse.
  6. Incubate for 10 min in the dark at room temperature.
  7. Measure the fluorescence at λex: 355 nm and λem: 420 nm in a microplate reader.

Data analysis

  1. Standards preparation for GSH and GSSG
    GSH and GSSG stock solution is prepared at a concentration of 1 mg/ml in KPE buffer and diluted 100 times to 10 µg/ml. 800 µl of 10 µg/ml solution is mixed with 200 µl KPE buffer to make standard GSH and GSSG concentration of 26.4 nmol/ml. From this concentration a series of two fold serial dilutions are (13.2 nmol/ml, 6.6 nmol/ml, 3.3 nmol/ml till 0.103 nmol/ml) prepared in KPE buffer.
  2. Calculation of protein concentration in unknown samples
    Protein concentrations is determined from standard curve with the formula Y = mX + c (Figures 4A and 4B).

    Figure 4. Calculation of protein concentration. A. Standard curve of BSA; B. Equation used for the calculation of protein concentration in an unknown sample.

  3. Calculation of GSH and GSSG in unknown samples
    Concentrations of GSH and GSSG from samples is calculated from their respective standard curve with the formula Y = mX + c (Figures 5A and 5B). This assay can detect GSH from 10 ng to 2 µg and GSSG from 5 ng to 2 µg.

    Figure 5. Calculation of GSH and GSSG concentration. Standard curve of (A) GSH; (B) GSSG and (C) Equation used for the calculation of GSH/GSSG concentration in an unknown sample.

  4. Normalization
    GSH and GSSG concentrations divided by the protein quantity used in reaction (10 µg protein per sample). Data are represented in terms of µmole/mg protein.


  1. 0.1 M potassium phosphate EDTA buffer (KPE buffer)
    Note: KPE buffer is prepared of two different solutions, A and B.
    Solution A: dissolving 0.68 g KH2PO4 in 50 ml dH2O
    Solution B: dissolving 0.85 g K2HPO4 or 1.14 g K2HPO4·3H2O in 50 ml dH2O
    Both the solution A and B can be stored at 4 °C if not using immediately. Just before use 0.1 M KPE buffer is prepared by mixing 8 ml of solution A with 42 ml of solution B and the pH adjusted to 8. Finally 0.16 g of EDTA disodium salt is added to the potassium phosphate buffer and mix well to prepare potassium phosphate EDTA buffer
  2. 50% trichloroacetic acid (10 ml)
    5 g trichloroacetic acid weighed and immediately mixed with dH2O
    Adjust the volume to 10ml with dH2O
  3. o-Phthaldehyde solution (10 mg/ml)
    o-Phthaldehyde solution is prepared by mixing 10 mg orthophthaldehyde with 1 ml methanol
    The solution can be stored at 4 °C in dark up to 1 week
  4. 0.4 M N-ethylmaleimide
    50 mg N-ethylmaleimide is dissolved in 1 ml 100% ethanol to make 0.4 M NEM solution. It can be stored at 4 °C up to 1 week
  5. 0.1 N sodium hydroxide
    0.04 g sodium hydroxide pellet is dissolved in 10 ml dH2O to get 0.1 N solution


This work has been supported by CSIR-12th 5 year network project, Integrated NextGen Approaches in Health, Disease and Environmental Toxicity (INDEPTH-BSC001); V.S. has been supported by CSIR Senior Research Fellowship. R.G. and M.P. P. have been supported by UGC-Senior Research Fellowship. The authors declare no competing financial interest. The CSIR-IITR manuscript number is 3440. This protocol has been followed and modified from paper entitled “A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74(1): 214-226”.


  1. Cohn, V. H and Lyle, J. (1966). A fluorometric assay for glutathione. Anal Biochem 14(3): 434-40.
  2. Hissin, P. J. and Hilf, R. (1976). A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 74(1): 214-26.
  3. Rahman, I., Kode, A. and Biswas, S. K. (2006). Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1(6): 3159-65.
  4. Singh, V., Gera, R., Kushwaha, R., Sharma, A. K., Patnaik, S. and Ghosh, D. (2016). Hijacking microglial glutathione by inorganic arsenic impels bystander death of immature neurons through extracellular cystine/glutamate imbalance. Sci Rep 6: 30601.


谷胱甘肽是细胞中存在的主要抗氧化防御成分之一。它主要作为还原型谷胱甘肽(GSH)存在并转化为氧化型谷胱甘肽(GSSG),同时还可以减少自由基,如羟基离子(OH)。对于GSH和GSSG的测定,邻苯二甲醛(OPT)已被用作荧光试剂。邻苯二甲醛具有与pH8的GSH和pH12的GSSG特异性反应的能力。已经使用N-甲基马来酰亚胺(NEM)来在本方案中测量GSSG期间防止GSH的自动氧化。 Hissin和Hilf的原始方案被开发用于大鼠肝组织中的谷胱甘肽估计。目前的方案已经在Hissin和Hilf(1976)之后被标准化,用于估计培养的小胶质细胞裂解物中的谷胱甘肽,但它也可以用于其它哺乳动物细胞裂解物。在我们的实验中,相同的方案已被用于估计小鼠神经母细胞瘤细胞N2a的全细胞裂解物中的谷胱甘肽。

背景 该方法于1976年由Hissin和Hilf在分析生物化学方面出版(Hissin和Hilf,1976)。然而,有可用于准确检测GSH的方法;由于GSH容易氧化转化为GSSG,所以大多数方法给出过高的GSSG。 Cohen和Lyle(1966)通过使用NEM来防止GSH氧化转化为GSSG并在GSSG估计过程中防止GSH与OPT反应(图1和图2)解决了这个问题。我们使用这种简单可靠的方法来检测我们的实验系统(小胶质细胞裂解物)中的GSH和GSSG。该协议的主要优点是,它不涉及复杂的仪器,如高效液相色谱(HPLC),与需要更常用和易于操作的读卡器相比,该液相色谱(HPLC)也需要足够的专业知识来处理(Rahman et al。 al。,2006)。

图1. N9细胞裂解物中GSH估计期间的化学反应示意图

图2. N9细胞裂解物中GSSG估计期间的化学反应示意图

关键字:谷胱甘肽, 抗氧化剂, 自由基, 小胶质细胞(N9), 邻苯二甲醛, N—乙基马来酰亚胺


  1. 移液器吸头(Corning,Axygen ®,目录号:T-1005-WB-C-L)
  2. 0.22μm过滤器
  3. 6培养皿(SRL LIFE SCIENCES,目录号:30006)
  4. 文化瓶
  5. 1.5ml离心管(Corning,Axygen ,目录号:MCT-150-R)
  6. 0.5ml管(Corning,Axygen ,目录号:14-222-292)
  7. 96孔板(SPL生命科学,目录号:30096)
  8. 一次性塑料电池刮刀(SRL LIFE SCIENCES,目录号:90020)
  9. 细胞系:在目前的方案中,小鼠胶质细胞系N9已被使用,由印度国家脑研究中心(NBRC)Anirban Basu博士(Singh等人,2016年) )。通过逆转录病毒转染原代小胶质细胞与禽类逆转录病毒MH2的v-myc或v-mil致癌基因开发N9细胞系。细胞在补充有10%FBS和1%青霉素 - 链霉素的DMEM / F12培养基中于37℃在5%CO 2培养箱中培养
  10. DMEM / F12(Sigma-Aldrich,目录号:56498C)
  11. 碳酸氢钠
  12. 双蒸水
  13. 胎牛血清(FBS)(Genetix Biotech,Cell clone TM,目录号:CCS-500-SA-U)
  14. 青霉素 - 链霉素(Pen-Strep)(Thermo Fisher Scientific,Gibco TM,目录号:10378016)
  15. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  16. Bradford试剂(Bio-Rad Laboratories,目录号:5000006)
  17. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
  18. 邻苯二甲醛(Sigma-Aldrich,目录号:P1378)
  19. 乙基马来酰亚胺(Sigma-Aldrich,目录号:E3876)
  20. 谷胱甘肽(GSH)(Sigma-Aldrich,目录号:G4251)
  21. 氧化谷胱甘肽(GSSG)(Sigma-Aldrich,目录号:G4376)
  22. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  23. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  24. 磷酸氢二钾(K 2 N 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  25. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)
  26. EDTA二钠盐(Sigma-Aldrich,目录号:E5513)
  27. 100%乙醇(Merck,目录号:1009831011)
  28. 甲醇(SRL实验室,目录号65524)
  29. 0.1M磷酸钾EDTA缓冲液(KPE缓冲液)(参见食谱)
  30. 50%三氯乙酸(见食谱)
  31. 邻苯二甲醛溶液(10mg / ml)(参见食谱)
  32. 0.4M乙基马来酰亚胺(参见食谱)
  33. 0.1 N氢氧化钠(见配方)


  1. 1 ml和200μl移液器(Eppendorf)
  2. 台式离心机(Sigma-Zentrifuges,型号:Sigma 3-18KS)
  3. 酶标仪(BMG LABTECH,型号:FLUOstar Omega)
  4. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific& TM),型号:Forma TM Steri-Cycle TM sub> 2 孵化器)
  5. 超声波仪(Sonics& Materials,型号:VC 505)


  1. 火星数据分析软件1.01(酶标仪数据分析软件)




  1. 媒体准备和细胞培养
    1. 对于1L培养基,将12g DMEM / F12,2.44g碳酸氢钠溶解在890ml双蒸水中,然后加入100ml FBS以及10ml Pen-Strep。最后,通过0.22μm过滤器过滤介质。
    2. 在6孔培养板中以1×10 6个/ ml的密度接种N9细胞,并在完全培养基(补充有10%FBS的DMEM / F12培养基)中生长16-18小时和1%Pen-Strep)。
    3. N9细胞每48小时传代,解冻后直至6-7代。
  2. 细胞收获和裂解
    1. 取出培养基并用磷酸盐缓冲盐水(PBS)洗涤细胞。向每个孔中加入冰冷的0.1M磷酸钾缓冲液(KPE缓冲液,1ml),借助于细胞刮板将细胞从培养瓶的表面移出,并收集在1.5ml离心管中。
    2. 在4℃将细胞悬浮液以350g离心5分钟。
    3. 将得到的细胞沉淀物重新悬浮于具有1%蛋白酶抑制剂混合物(PIC)的200μlKPE缓冲液中,并通过超声处理5秒脉冲在25W下在冰上裂解两次。
  3. 蛋白质样品制备和评估
    将10μl样品保存在0.5 ml管中,用Bradford试剂进行蛋白质评估。
  4. 蛋白沉淀
  5. GSH和GSSG估计
    1. GSH估计
      将10μl上清液与黑色96孔板中的等体积的OPT(1mg / ml)和180μlKPE缓冲液(pH-8)混合。
    2. GSSG估计
      1. 将50μl上清液转移到新的离心管中,加入0.5μlN-乙基马来酰亚胺(储备浓度:4M),并充分混合。
      2. 在室温下孵育30分钟以抑制GSH。
      3. 在黑色96孔板中加入10μl该样品,10μlOPT和180μl0.1N NaOH(pH-12)。
  6. 在室温下在黑暗中孵育10分钟。
  7. 在酶标仪中测量λ<λ> 355nm和λ 420nm处的荧光。


  1. 标准准备GSH和GSSG
    在KPE缓冲液中以1mg / ml的浓度制备GSH和GSSG储备溶液,并稀释100倍至10μg/ ml。将800μl的10μg/ ml溶液与200μlKPE缓冲液混合,使标准GSH和GSSG浓度为26.4nmol / ml。从该浓度,在KPE缓冲液中制备一系列两倍连续稀释度为(13.2nmol / ml,6.6nmol / ml,3.3nmol / ml至0.103nmol / ml)。
  2. 计算未知样品中的蛋白质浓度
    蛋白质浓度由公式Y = mX + c的标准曲线确定(图4A和4B)。

    图4.蛋白质浓度的计算 A. BSA的标准曲线; B.用于计算未知样品中蛋白质浓度的方程。

  3. 计算未知样品中的GSH和GSSG
    来自样品的GSH和GSSG的浓度由其各自的标准曲线由式Y = mX + c计算(图5A和5B)。该测定可以检测10ng至2μg的GSH和5ng至2μg的GSSG

    图5. GSH和GSSG浓度的计算(A)GSH的标准曲线; (B)GSSG和(C)用于计算未知样品中GSH / GSSG浓度的方程式。

  4. 规范化
    GSH和GSSG浓度除以反应中使用的蛋白质量(每个样品10μg蛋白质)。数据以μmole/ mg蛋白表示。


  1. 0.1M磷酸钾EDTA缓冲液(KPE缓冲液)
    溶液A:将0.68g KH 2 PO 4在50ml dH 2 O中溶解
    溶液B:溶解0.85克K 2 HPO 4或1.14克K 2 HPO 4·3H 2 O 2在50ml dH 2 O中的溶液 如果不立即使用,溶液A和B都可以在4℃下储存。使用前0.1M KPE缓冲液通过将8ml溶液A与42ml溶液B混合并将pH调节至8来制备。最后将0.16g EDTA二钠盐加入到磷酸钾缓冲液中并充分混合以制备磷酸钾EDTA缓冲液
  2. 50%三氯乙酸(10ml)
    称量5g三氯乙酸,并立即与dH 2 O immediately immediately混合 用dH <2> O
  3. 邻苯二甲醛溶液(10mg / ml)
    通过将10mg邻苯二甲醛与1ml甲醇混合来制备邻苯二甲醛溶液 溶液可以在黑暗中保存在4°C至1周
  4. 0.4M N - 乙基马来酰亚胺
    将50mg N-乙基马来酰亚胺溶于1ml 100%乙醇中以制备0.4M NEM溶液。它可以在4°C存储1个星期
  5. 0.1N氢氧化钠
    将0.04g氢氧化钠颗粒溶解在10ml dH 2 O中以得到0.1N溶液


这项工作得到CSIR-12th 5年网络项目“健康,疾病与环境毒性综合NextGen方法”(INDEPTH-BSC001)的支持。 V.S.获得CSIR高级研究奖学金的支持。 R.G.和M.P.教资会资助高级研究奖学金。作者声称没有竞争的经济利益。 CSIR-IITR手稿编号为3440.该方案已经从题为“用于测定组织中氧化和还原型谷胱甘肽的荧光测定方法”的文献得到遵循和修改。 Anal Biochem 74(1):214-226“。


  1. Cohn,V.H和Lyle,J.(1966)。&lt; a class =“ke-insertfile”href =“http://www.sciencedirect.com/science/article/pii/0003269766902867”target =“_ blank “>用于谷胱甘肽的荧光测定法。 14(3):434-40。
  2. Hissin,PJ和Hilf,R。(1976)。&nbsp; 用于测定组织中氧化和还原型谷胱甘肽的荧光测定法。。 74(1):214-26。
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引用:Singh, V., Gera, R., Purohit, M. P., Patnaik, S. and Ghosh, D. (2017). Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate. Bio-protocol 7(11): e2304. DOI: 10.21769/BioProtoc.2304.