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Cryopreservation Protocol for Chlamydomonas reinhardtii
莱茵衣藻的冷冻保存方案   

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Abstract

Cryopreservation is commonly used for storing viable cells, tissues, organs or organisms at ultralow temperatures, and usually involves immersion in liquid nitrogen at -196 °C. Here we provide a detailed cryopreservation protocol for C. reinhardtii based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes (Yang and Li, 2016). In this study, we compared the cryoprotection effect of two common cryopreservation agents (CPAs), methanol and DMSO. Furthermore, the two-step cryopreservation process was divided into five stages to study the factors affecting the survival rate at each stage. We found that the use of methanol as the CPA, combined with the cooling process outlined here (cooling from 25 °C to -55 °C at a rate of 1 °C/min), were indispensable for cell survival after cryopreservation. The thawing process described here (thawing at 35 °C for 5 min) was also important for increasing the survival rate.

Background

Nowadays, cryopreservation is used frequently for the storage of transgenic lines or mutation lines of C. reinhardtii, and for experimental needs involving this organism. Morris et al. (1979) discussed the effects of different CPAs in cooling, the relationships between temperature and survival rate with or without the CPAs, and the cooling rate. They found that with the addition of methanol the half-lethal temperature was the lowest of all the CPAs tested (-14.4 °C), while that of DMSO was -4.9 °C (Morris et al., 1979).

Through storage in 7% (v/v) DMSO overnight at room temperature, followed by storage at -70 °C, Johnson and Dutcher (1993) gained the highest viabilities, nearly 10% in C. reinhardtii cultures. However, the survival rate may be restricted in C. reinhardtii cell lines, especially in the cell line CC-125 we used, with viabilities of only 0.34%, although the authors claim was caused by liquid culturing. Nevertheless, this method was time consuming and resulted in low cell viabilities. Crutchfield et al. (1999) reported a two-step cooling procedure for the cryopreservation of C. reinhardtii using 5% methanol as the CPA, which retained relatively high viability (> 40%).

Three different methods for improving survival rate were compared in this study. We followed the protocols mentioned above, and made detailed analyses at each stage of the cryopreservation process. The different effects of methanol and DMSO are also discussed, the results agreeing with the work of previous authors.

Materials and Reagents

  1. 1.5 ml and 4 ml Eppendorf tubes
  2. 50 ml flask
  3. Cryovials (VWR, Nalgene®, catalog number: 5000 )
  4. Filter membrane (Filter pore size: 0.2-0.3 μm) for tissue culture
  5. 0.22 μm membrane filters (EMD Millipore, model: SLGV033RB )
  6. Chlamydomonas reinhardtii CC125 was purchased from the Chlamydomonas Resource Center at the University of Minnesota (http://www.chlamycollection.org/cart/)
  7. TAP medium (Gorman and Levine, 1965, http://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/)
  8. Isopropanol, purity ≥ 99.5% (Sangon Biotech, catalog number: A507048 )
  9. Liquid nitrogen
  10. Trypan blue (Sigma-Aldrich, catalog number: T6146 )
  11. Methanol (MeOH) (AR), purity ≥ 99% (Chongqing Chuandong Chemical, catalog number: methanol )
  12. Dimethyl sulfoxide (DMSO) (Sangon Biotech, catalog number: A503039 )
  13. Iodine (AR) (Sangon Biotech, catalog number: A500538 )
  14. Potassium iodide (AR) (Sangon Biotech, catalog number: A100512 )
  15. Lugol’s iodine solution (see Recipes)
  16. CPA stock solution (see Recipes)

Equipment

  1. Thermostatic rocking incubator (Shanghai Shipping, model: SPH-211B )
  2. Hemocytometer (Qiujing)
  3. Freezing container (Cryo 1 °C freezing container) (VWR, Nalgene®, model: 5100 )
  4. Autoclave
  5. Laminar flow hood
  6. Microscope (OLYMPUS, model: CX31 )
  7. Water bath (Amersham Bioscience)
  8. Portable liquid nitrogen tank
  9. Centrifuge (Hettich, model: D-78532 Tuttlingen)
    Note: This equipment has been discontinued, other types with the same centrifugal force can be used as substitutions.
  10. -80 °C freezer (Thermo Fisher Scientific, model: 8607 )

Software

  1. Microsoft Office Excel

Procedure

  1. Cell culture conditions
    1. Cell culture is grown using 20 ml of TAP medium in a 50 ml flask. 1 ml of stationary-phase cell culture is transferred into 20 ml fresh medium every week. The culture conditions are adjusted to a speed of 120 rpm shaking at 25 °C using a Thermostatic rocking incubator, and the culture is illuminated continuously with white fluorescent light (PAR = 30 μmol photons m-2 s-1). Cultures at a cell density of approximately 0.9-2.0 x 107 ml-1 (3-5 days after transfer, late linear phase) are used for the experiment.

  2. Calculating the cell density of C. reinhardtii and volume of cell culture needed in a 2 ml cryovial
    1. 3-5 d after transfer, take 1 ml cell culture out of the 50 ml flask for cell density estimating.
    2. Measure cell density using a hemocytometer: mix 10% volume (10 μl) of Lugol’s iodine solution with 90 μl cell culture (diluted if necessary) and add 8 μl of the mixture to each chamber.
    3. Count five small squares in each chamber as a means of calculating the mean number of cells per chamber. The cell number per microliter = mean numbers of cells in five small squares x 5 x 104 x dilution factor (10/9, if not further diluted). Optimally, the number of cells should be approximately 0.9-2.0 x 107 cells per ml.
      Note: Steps B2 and B3 can be substituted by other cell counting methods.
    4. Calculate the volume of cell culture needed in a 2 ml cryovial and confirm that the final density in the cryovial is no more than 3.3 x 106 cells per ml (it was exactly 3.3 x 106 in our experiment). It has been reported that when the final cell density in a cryovial is more than 3.3 x 106 cells per ml, the survival rate after cryopreservation decreases significantly (Piasecki et al., 2009). Cell culture volume in 2 ml cryovial = 3.3 x 106 cells per ml x 1.8 ml/the number of cells per ml. Supplement with TAP medium at: volume of TAP medium = 0.9 ml-cell culture volume.

  3. Preparation and conditions for the cryopreservation process
    1. Precool the CPA stock solution to 4 °C.
    2. Put 250 ml isopropanol in the lower department of the two-compartment freezing container, precool it to 4 °C (chill for more than 30 min).
    3. Sterilize all the equipment and tubes used by ultraviolet sterilization and autoclave.
    4. Carry out all further operations for the further cultivation of C. reinhardtii cells in a clean and sterile environment (we suggest using a laminar flow hood) to avoid contamination.
    5. Avoid exposing the CPA (or culture containing CPA) to bright light, because methanol becomes toxic to cells after illumination (Crutchfield et al., 1999).
    Note: C. reinhardtii stains CC124, CC3395, CC1690 (http://www.chlamycollection.org) were cryopreserved by this protocol, which works well too.

  4. The cryopreservation process
    1. The cryopreservation process was based on Crutchfield’s work (Crutchfield et al., 1999), with minor changes. We divided the process into five stages, shown in Figure 1. The treatment 4 CC’LTR in Figure 1 is a typical cryopreservation process in this field.


      Figure 1. Cryogenic treatments and cryopreservation processes of C. reinhardtii. The process was divided into five stages: (i) Control (normal growth) (C); (ii) Cooling from 25 °C to -55 °C at a rate of 1 °C/min (C’); (iii) Freezing in liquid nitrogen (L); (iv) Thawing at 35 °C for 5 min (T); and (v) Recovery growth for 2 days under normal growth conditions (R). The processes that the organism underwent in each treatment is indicated by a solid line; for an omitted step, a dotted line is used. There were six different treatment combinations: (1) C: Control (normal growth) only; (2) CC’T: Control, Cooling and Thawing; (3) CC’LT: Control, Cooling, Freezing in liquid nitrogen (LN) and Thawing; (4) CC’LTR: Control, Cooling, Freezing in LN, Thawing and Recovery growth; (5) CLT: Control, Freezing in LN and Thawing; (6) CC’LR: Control, Cooling, Freezing in LN, Incubation at room temperature for 30 min and Recovery growth. (Yang and Li, 2016)

    2. Put 0.9 ml CPA stock solution (we used two other CPA controls to compare with methanol in our experiment, Table 1) and a corresponding volume (see step B4) of cell culture and TAP into a 2 ml cryovial, close the cap, mix gently. The total liquid volume in a 2 ml cryovial should be 1.8 ml and the final cell density 6.6 x 106 cells per ml.
    3. Put the cryovials into the upper compartment of the freezing container. Close the container and place it in a freezer at -80 °C, leaving it undisturbed for 1.5 h (the ‘Cooling’ phase in Figure 1).
    4. Remove the cryovials from the freezing container, put them into liquid nitrogen immediately, store for 1 day (‘Freezing in LN’ in Figure 1).
      Note: In our cryopreservation practice, C. reinhardtii survived in storage for at least 6 months.
    5. Remove the cryovials from the liquid nitrogen, then quickly transfer them to a water bath at 35 °C for 5 min (‘Thawing’ in Figure 1).
    6. Centrifuge the cryovials at 1,000 x g for 2 min. Then discard the liquid supernatant, add 1 ml of fresh TAP to the cryovials for culturing (preventing contamination). Loosen the cap of the cryovials, mix their contents twice a day. The culture conditions should be the same as in the Control phase, but without shaking (‘Recovery’ in Figure 1).

  5. Calculation of survival rate
    To assess the integrity of the cell membranes, the Evans blue dye test was used. The viability of cultures after cryopreservation is reflected by the percentage of cells that excluded the Evans blue dye. The method follows Crutchfield et al. (1999) with minor changes.
    1. Mix equal volumes of Evans blue dye (0.1% w/v in water) and cell culture (diluted if needed), leave undisturbed in an Eppendorf tube for 5 min.
    2. Add about 8 μl mixture to each chamber of a hemocytometer to enable counting under a microscope. At least 200 cells were examined at 400x magnification in a chamber, both chambers were counted independently to determine whether the cells had taken up the Evans blue dye.
    3. Calculate the survival rate as follows: the ratio of the number of cells that retained their original green color to the total number of cells. Each of the treatments outlined above was performed five times. The survival rate for each treatment (Figure 1) in our experiment is shown in Table 1.
    Note: The Evans blue dye method works well when counting cells immediately after thawing. After further culturing or a long time after thawing, the alive cells migrate fast, which makes them difficult to count.

Table 1. Survival rate of C. reinhardtii in culture medium, culture medium plus 5% DMSO, and culture medium plus 5% MeOH after cryogenic treatments (Yang and Li, 2016)

CM, culture medium; CM + DMSO, culture medium plus 5% DMSO; CM + MeOH, culture medium plus 5% MeOH. Values are means ± standard deviation (n = 5). ‘*’ represents those samples in which the survival rate was calculated immediately after thawing without further culturing.

Data analysis

The survival of C. reinhardtii after cryopreservation was assessed by Evan’s blue method (see Procedure E) (Crutchfield et al., 1999). For each treatment (2-6) in Table 1, five samples were counted. The mean and standard deviation was calculated by functions ‘AVERAGE’ and ‘STDEV’ respectively in Microsoft Office Excel.

Notes

  1. These experiments were repeated three times on different dates (2014.7, 2015.5 and 2015.6), the survival rate in CC’LT treatment being 55.38%, 34.6%, 41.3%, respectively. The second time, the CPAs used had been stored at 4 °C for more than three months, so if fresh CPAs are used, the viability seems to rise. Estimation by eye under a light microscope of whether or not a cell was stained with Evan’s blue will introduce some random errors.
  2. Additional fluorescent lighting will be needed in the Thermostatic rocking incubator to reach the required illumination intensity.
  3. Make sure the C. reinhardtii cells are well cultured: the liquid should be yellow-green or green and uniform, running under the microscope, and mainly comprising single cells, with no aggregates. We used the cells 3-5 days after transfer from liquid culture. If cultured from cryopreservation, 2 or more liquid transfers may be necessary.
  4. Avoid light in the steps with CPAs. The CPAs stocks should be stored at 4 °C, for no longer than 1 month.
  5. Please handle liquid nitrogen and the -80 °C freezer with care to avoid injury.

Recipes

  1. Lugol’s iodine solution
    1 g I2, 2 g KI, with distilled water added to total volume of 20 ml
    Dilute 10-fold when used
  2. CPA stock solution
    CPA stock solution: 10% MeOH in TAP medium
    Control: TAP only or 10% DMSO in TAP medium

Acknowledgments

This protocol was adapted from previously published study of Crutchfield et al. (1999). Our thanks also go to Matt Laudon, curator of the Chlamydomonas Resource Center, who provided the cell lines. This work was performed by Yang and Li (2016). The author thanks Dr. Jane Marczewski, Zheng Guowei, Chen Hongying for critical reading the protocol. This research was supported by grants from the National Natural Science Foundation of China (31070262), Kunming Institute of Botany (KSCX2-EW-J-24), the Germplasm Bank of Wild Species and the CAS Innovation Program of Kunming Institute (540806321211), as well as the 100-Talents Program of CAS.

References

  1. Crutchfield, A. L. M., Diller, K. R. and Brand, J. J. (1999). Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). Eur J Phys 34(1): 43-52.
  2. Gorman, D. S. and Levine, R. P. (1965). Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A 54(6): 1665-1669.
  3. Johnson, D. E. and Dutcher, S. K. (1993). A simple, reliable method for prolonged frozen storage of Chlamydomonas. Trends Genet 9(6): 194-195.
  4. Morris, G. J., Coulson, G. and Clarke, A. (1979). The cryopreservation of Chlamydomonas. Cryobiology 16(4): 401-410.
  5. Piasecki, B. P., Diller, K. R. and Brand, J. J. (2009). Cryopreservation of Chlamydomonas reinhardtii: a cause of low viability at high cell density. Cryobiology 58(1): 103-109.
  6. Yang, D. and Li, W. (2016). Methanol-promoted lipid remodelling during cooling sustains cryopreservation survival of Chlamydomonas reinhardtii. PLoS One 11(1): e0146255.

简介

冷冻保存通常用于在超低温度下储存活细胞,组织,器官或生物体,并且通常包括在-196℃的液氮中浸泡。在这里,我们提供了一个详细的cryopreservation协议。基于Crutchfield的工作(Crutchfield et al 。,1999),并进行微小的改变(Yang和Li,2016)。在这项研究中,我们比较了两种普通冷冻保存剂(CPAs),甲醇和DMSO的冷冻保护作用。此外,两步冷冻保存过程分为五个阶段,以研究影响每个阶段的生存率的因素。我们发现使用甲醇作为CPA,结合此处概述的冷却过程(以1℃/min的速率从25℃冷却至-55℃)对于冷冻保存后的细胞存活是不可缺少的。这里描述的解冻过程(在35℃解冻5分钟)对于提高存活率也是重要的。

[背景] 现在,冷冻保存经常用于储存C的转基因品系或突变系。以及涉及该生物体的实验需要。 Morris等人。 (1979)讨论了不同CPA在冷却中的影响,温度和存活率之间的关系,有或没有CPA,和冷却速度。他们发现,加入甲醇后,半致死温度是测试的所有CPA中最低的(-14.4℃),而DMSO的温度为-4.9℃(Morris等人, 1979)。
 通过在室温下在7%(v/v)DMSO中储存过夜,随后在-70℃下储存,Johnson和Dutcher(1993)获得了最高的活力,在10℃下接近10%。莱茵哈德文化。然而,存活率可以在C中被限制。特别是在我们使用的细胞系CC-125中,尽管作者的主张是由液体培养引起的,但存活率仅为0.34%。然而,该方法是耗时的并且导致低的细胞存活力。 Crutchfield 。 (1999)报道了C的冷冻保存的两步冷却程序。使用5%甲醇作为CPA,其保持相对高的活力(> 40%)。
 在本研究中比较了改善存活率的三种不同方法。我们遵循上述方案,并在冷冻保存过程的每个阶段进行详细分析。也讨论了甲醇和DMSO的不同影响,结果与以前的作者的工作一致。

材料和试剂

  1. 1.5 ml和4 ml Eppendorf管
  2. 50ml烧瓶中
  3. 冷冻瓶(VWR,Nalgene ,目录号:5000)
  4. 用于组织培养的滤膜(过滤器孔径:0.2-0.3μm)
  5. 0.22μm膜过滤器(EMD Millipore,型号:SLGV033RB)
  6. CC125购自明尼苏达大学的Chlamydomonas资源中心( http://www.chlamycollection.org/cart/
  7. TAP培养基(Gorman和Levine,1965, 设备

    1. 恒温摇摆培养箱(上海航运,型号:SPH-211B)
    2. 血细胞计数器(Qiujing)
    3. 冷冻容器(Cryo 1℃冷冻容器)(VWR,Nalgene ,型号:5100)
    4. 高压灭菌器
    5. 层流罩
    6. 显微镜(OLYMPUS,型号:CX31)
    7. 水浴(Amersham Bioscience)
    8. 便携式液氮罐
    9. 离心机(Hettich,型号:D-78532 Tuttingen)
      注意:此设备已停产,其他类型的离心力相同可用作替代。
    10. -80℃冷冻器(Thermo Fisher Scientific,型号:8607)

    软件

    1. Microsoft Office Excel

    程序

    1. 细胞培养条件
      1. 使用20ml的TAP培养基在50ml烧瓶中培养细胞培养物。每周将1ml固定相细胞培养物转移到20ml新鲜培养基中。使用恒温摇动培养箱将培养条件在25℃下调节至120rpm的速度,并且用白色荧光灯(PAR =30μmol光子m <-200s -1)连续照射培养物, sup> -1 )。以约0.9-2.0×10 7个细胞密度的细胞密度的培养物(转移后3-5天,晚期线性期)用于实验。

    2. 计算C的细胞密度。和在2ml冷冻管中所需的细胞培养物体积
      1. 3-5天后转移,取1 ml细胞培养物从50ml烧瓶中进行细胞密度估算。
      2. 使用血细胞计数器测量细胞密度:将10%体积(10μl)的Lugol碘溶液与90μl细胞培养物(如果需要稀释)混合,并将8μl混合物加入每个室。
      3. 计算每个室中的五个小正方形作为计算每个室的平均细胞数的方法。每微升的细胞数=五个小方块中的平均细胞数×5×10 4稀释倍数(10/9,如果不进一步稀释)。最佳地,细胞的数量应该为约0.9-2.0×10 7个/ml细胞/ml。
        注意:步骤B2和B3可以替换为其他细胞计数方法。
      4. 计算在2ml冷冻管中所需的细胞培养物的体积,并确认冷冻管中的最终密度不超过3.3×10 6个细胞/ml(其恰好是3.3×10 6 )。已经报道,当冷冻管中的最终细胞密度大于3.3×10 6个细胞/ml时,冷冻保存后的存活率显着降低(Piasecki等人)。 ,2009)。 2ml冷冻保护的细胞培养体积= 3.3×10 6个细胞/ml×1.8ml /每毫升的细胞数。用TAP培养基补充:TAP培养基体积= 0.9ml-细胞培养物体积
    3. 冷冻保存过程的准备和条件
      1. 将CPA储备溶液预冷至4°C
      2. 将250 ml异丙醇放入双室冷冻容器的下部,预冷至4°C(冷却30分钟以上)。
      3. 消毒紫外线杀菌和高压灭菌器所用的所有设备和管
      4. 进行进一步培养C的所有进一步操作。 (我们建议使用层流罩),以避免污染。
      5. 避免将CPA(或含有CPA的培养物)暴露于明亮的光下,因为甲醇在照射后对细胞有毒性(Crutchfield等人,1999)。
      注意:C. reinhardtii污渍CC124,CC3395,CC1690(
      http://www.chlamycollection.org )通过此协议冻存,这也很好。

    4. 冷冻保存过程
      1. 冷冻保存过程基于Crutchfield的工作(Crutchfield等人,1999),具有微小的变化。我们将过程分为五个阶段,如图1所示。图1中的处理4 CC'LTR是该领域中的典型的低温保存过程。



        图1. C的低温处理和冷冻保存过程。 。该过程分为五个阶段:(i)控制(正常生长)(C); (ii)以1℃/min(℃)的速率从25℃冷却至-55℃; (iii)在液氮中冷冻(L); (iv)在35℃解冻5分钟(T);和(v)在正常生长条件(R)下恢复生长2天。生物体在每次处理中进行的过程用实线表示;对于省略的步骤,使用虚线。有6种不同的治疗组合:(1)C:仅控制(正常生长) (2)CC'T:控制,冷却和解冻; (3)CC'LT:控制,冷却,在液氮(LN)冷冻和解冻; (4)CC'LTR:控制,冷却,冻结LN,解冻和恢复生长; (5)CLT:对照,LN冻结和解冻; (6)CC'LR:控制,冷却,在LN中冷冻,在室温下孵育30分钟并恢复生长。 (Yang and Li,2016)

      2. 将0.9ml CPA储备溶液(我们在我们的实验中使用两种其他CPA对照与甲醇进行比较,表1)和细胞培养物和TAP的相应体积(参见步骤B4)放入2ml冷冻管中,盖上盖子,轻轻混合。 2ml冷冻管中的总液体体积应为1.8ml,最终细胞密度为6.6×10 6个/ml细胞。
      3. 将冷冻管放入冷冻容器的上部隔室。关闭容器,将其置于-80℃的冰箱中,使其不受干扰1.5小时(图1中的"冷却"阶段)。
      4. 从冷冻容器中取出冷冻管,立即将其置于液氮中,储存1天(图1中的"冻结LN")。
        注意:在我们的冷冻保存实践中,C. reinhardtii在存储中存活至少6个月。
      5. 从液氮中取出冷冻管,然后迅速将其转移到35℃的水浴中5分钟(图1中的"解冻")。
      6. 在1,000×g离心冷冻小瓶2分钟。然后丢弃液体上清液,加入1ml新鲜TAP到冷冻管用于培养(防止污染)。松开冷冻瓶的盖子,每天混合其内容两次。培养条件应与对照阶段相同,但不摇动(图1中的"恢复")
    5. 存活率的计算
      为了评估细胞膜的完整性,使用伊文思蓝染料试验。冷冻保存后培养物的存活率由排除Evans蓝色染料的细胞的百分比反映。该方法遵循Crutchfield et al。(1999),但有轻微的变化
      1. 混合等体积的伊文思蓝染料(0.1%w/v的水溶液)和细胞培养物(如果需要稀释),在Eppendorf管中静置5分钟。
      2. 向血细胞计数器的每个室中加入约8μl混合物以使得能够在显微镜下计数。在室中以400×放大倍数检查至少200个细胞,独立地计数两个室以确定细胞是否摄取了伊文思蓝染料。
      3. 计算存活率如下:保留其原始绿色的细胞数量与细胞总数的比率。上述每种处理进行5次。我们的实验中每种处理的存活率(图1)显示在表1中。
      注意:在解冻后立即计数细胞时,伊文思蓝染料方法工作得很好。在进一步培养或解冻后长时间后,活细胞快速迁移,这使得它们难以计数。

    表1. C的存活率 。 在培养基,培养基加5%DMSO和培养基加5%MeOH的冷冻干燥之后(杨和李,2016)
    CM,培养基; CM + DMSO,培养基加5%DMSO; CM + MeOH,培养基加5%MeOH。值为平均值±标准偏差(n = 5)。 "*"表示其中在解冻后立即计算存活率而无需进一步培养的那些样品。

    数据分析

    C的存活。通过Evan的蓝色方法(参见程序E)(Crutchfield ,1999)。对于表1中的每次处理(2-6),计数5个样品。在Microsoft Office Excel中分别通过函数"AVERAGE"和"STDEV"计算平均值和标准偏差。

    笔记

    1. 这些实验在不同的日期(2014.7,2015.5和2015.6)重复三次,CC'LT治疗的存活率分别为55.38%,34.6%,41.3%。第二次,所用的CPA已在4℃下储存超过三个月,因此如果使用新鲜的CPA,则活力似乎上升。在光学显微镜下通过眼睛估计细胞是否用伊文蓝染色将引入一些随机误差。
    2. 在恒温摇摆培养箱中需要额外的荧光照明以达到所需的照明强度
    3. 确保 > 细胞培养良好:液体应该是黄绿色或绿色和均匀的,在显微镜下运行,主要包括单细胞,没有聚集体。我们使用从液体培养转移后3-5天的细胞。如果从冷冻保存培养,可能需要2次或更多次液体转移
    4. 在CPA的步骤中避免光线。 CPA库存应在4°C储存不超过1个月。
    5. 请小心处理液氮和-80°C冰箱,以免受伤。

    食谱

    1. Lugol的碘溶液
      1g I 2,2g KI,加入蒸馏水至总体积为20ml。
      使用时稀释10倍
    2. CPA储存溶液
      CPA储备溶液:10%MeOH/TAP介质
      对照:在TAP培养基中仅含TAP或10%DMSO,

    致谢

    此协议改编自之前发表的Crutchfield 。 (1999)。我们还感谢提供细胞系的Chlamydomonas资源中心的策展人Matt Laudon。这项工作是由杨和李(2016年)。作者感谢简Marczewski博士,郑国维,陈红英批评阅读协议。本研究得到国家自然科学基金(31070262),昆明植物研究所(KSCX2-EW-J-24),野生种质种质库和昆明研究院CAS创新项目(540806321211)资助,以及CAS的100人才计划。

    参考文献

    1. Crutchfield,ALM,Diller,KR和Brand,JJ(1999)。  reinhardtii (Chlorophyta)的冷冻保存。/em> J Phys 34(1):43-52。
    2. Gorman,DS和Levine,RP(1965)。  细胞色素f和质体蓝素:它们在Chlamydomo nas reinhardi的光合电子传递链中的序列。
    3. Johnson,DE和Dutcher,SK(1993)。  A简单,可靠的方法,用于长时间冷冻储存 amydomonas。 9(6):194-195。
    4. Morris,GJ,Coulson,G.和Clarke,A。(1979)。  401-410。
    5. Piasecki,BP,Diller,KRand Brand,JJ(2009)。  Chlamydo 的冷冻保存:高细胞密度下存活率低的原因 Cryob em> 58(1):103-109
    6. Yang,D。和Li,W.(2016)。  甲醇促进的脂质重塑在冷却过程中维持衣原体 的冷冻保存。 em> O 11(1):e0146255。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Yang, D. and Li, W. (2016). Cryopreservation Protocol for Chlamydomonas reinhardtii. Bio-protocol 6(22): e2024. DOI: 10.21769/BioProtoc.2024.
  2. Yang, D. and Li, W. (2016). Methanol-promoted lipid remodelling during cooling sustains cryopreservation survival of Chlamydomonas reinhardtii. PLoS One 11(1): e0146255.
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