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The protocol is very reliable and simple for inducing heat shock in unicellular green algae cells. The main purpose was to compare cellular response of three Chlorella species, isolated from different habitats: Chlorella vulgaris 8/1- thermophilic, Chlorella kesslery- mesophilic and C. vulgaris- extremophilic. Species were isolated from different habitats and differ in their temperature preferences and tolerance. Temperature induced stress response was measured as cell survival, induction of chloroplast HSP70B and DSBs induction and rejoining.

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Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells

Plant Science > Phycology > Cell analysis
Authors: Stephanie Chankova
Stephanie ChankovaAffiliation: Ecosystem Research, Ecological Risk Assessment And Conservation Biology, Institute of Biodiversity and Ecosystem Research, BAS, Sofia, Bulgaria
For correspondence: stephanie.chankova@yahoo.com
Bio-protocol author page: a396
Zhana Mitrovska
Zhana MitrovskaAffiliation: Ecosystem Research, Ecological Risk Assessment And Conservation Biology, Institute of Biodiversity and Ecosystem Research, BAS, Sofia, Bulgaria
Bio-protocol author page: a737
 and Nadezhda Yurina
Nadezhda YurinaAffiliation: Laboratory of Bioenergetics, A.N. Bach Institute of Biochemistry RAS, Moscow, Russia
For correspondence: nyurina@inbi.ras.ru
Bio-protocol author page: a397
Vol 3, Iss 15, 8/5/2013, 2503 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.849

[Abstract] The protocol is very reliable and simple for inducing heat shock in unicellular green algae cells. The main purpose was to compare cellular response of three Chlorella species, isolated from different habitats: Chlorella vulgaris 8/1- thermophilic, Chlorella kesslery- mesophilic and C. vulgaris- extremophilic. Species were isolated from different habitats and differ in their temperature preferences and tolerance. Temperature induced stress response was measured as cell survival, induction of chloroplast HSP70B and DSBs induction and rejoining.

Materials and Reagents

  1. Species:
    Three Chlorella species were used: C. vulgaris, isolated from soil samples of Livingston Island, the South Shetland Archipelago, Antarctic; C. vulgaris strain 8/1, isolated in 1968 from thermal springs in the region of Rupite, Bulgaria, and cultivated in our laboratory since 1975 and Chlorella kesslery a mesophile, from the Trebon collection.
    Cultivation: Chlorella species were cultivated on Tris Acetate Phosphate (TAP) medium under continuous light of 60 μmol/m2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber. The species were cultivated at this temperature because it is well known, that eurythermal algae, could be grown at a wide range of temperatures.
  2. TAP medium (see Recipes)
  3. Sager–Granick medium (see Recipes)

Equipment

  1. Phytotron GC 400 growth chamber (NUVE Ankara/Turkey 2009)
  2. Microscope
  3. Bürker chamber
  4. Circulating water bath

Procedure

  1. Cultivation
    1. Cultivate for 4-5 days algae strains or species on TAP medium (Harris, 1989) under continuous light of 60 μmol/m2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber to the end of exponential and early stationary phase of growth.
    2. Check under the microscope whether cell culture is not contaminated.
    3. Count cell number under the microscope using Bürker chamber.
    4. Centrifuge certain volume (depending on the cell density, counted in step A-3) of cell suspension at 1,200 x g by RT, resuspend in Sager-Granick medium or other appropriate medium, so to get 10 ml of cell suspension with a density of 1 x 106 cells/ml for every sample.

  2. Temperature treatment
    1. Keep 10 ml cell culture with a density 1 x 106 cells/ml in an incubator under continuous shaking, at different temperatures:
      t = 39 °C for 30 min
      t = 42 °С for 5 min
      t = 45 °С for 5 min
      Notes:
      1. The same heating procedure could be done in a circulating water bath at the same three temperatures: t = 39 °С for 30 min, t = 42 °С for 5 min, and t = 45 °С for 5 min.
      2. Use 50 ml flasks to comply with the requirement that volume of the cell culture must be not more than 1/3 of the volume of the flask.
    2. Place on ice to stop the heating process.
    3. For HSP70B analysis keep cells for 2 and 4 h after the step II-2 at t = 23 °C ± 0.1 °C to allow cells to recover.
    4. Centrifuge 10 ml cell suspension at 1,200 x g for 5 min.

Recipes

  1. TAP medium
    Stock solution for 1 L of TAP media
    1 M Tris base
    20 ml
    Phosphate Buffer II (see 1-a)
    1.0 ml
    Solution A (see 1-b)
    10.0 ml
    Hutner's trace elements (see 1-c)
    1.0 ml
    Glacial acetic acid (pH to 7.0)
    1.0 ml
    1. Phosphate buffer II (Stock solution)
      Component (For 100 ml)
      K2HPO4
      10.8 g
      KH2PO4
      5.6 g
    2. Solution A
      Component (For 500 ml)
      NH4Cl
      20 g
      MgSO4.7H2O
      5.0 g
      CaCl2.2H2O
      2.5 g
    3. Hutner's trace elements
      1. Dissolve 50 g of acid free EDTA in 250 ml of deionized. Heat to dissolve.
      2. Dissolve the following one by one in order. Heating to approximately 100 °C in 500 ml deionized H2O.
        Component
        Quantity
        H3BO3
        11.4 g
        ZnSO4.7H2O
        22.0 g
        MnCl2.4H2O
        5.06 g
        FeSO4.7H2O
        4.99 g
        CoCl2.6H2O
        1.61 g
        CuSO4.5H2O
        1.57 g
        Mo7O24(NH4)6.4H2O
        1.1 g
      3. Mix the two solutions together. The resulting solution should be blue-green.
      4. Heat to 100 °C. Cool slightly, but don't let the temperature drop below 80 °C – 90 °C.
      5. Adjust pH to 6.5–6.8 with 20% KOH (approximately 83 ml).
  2. Sager and Granick medium (adjust pH 6.8-7.0)
    Component
    In 1 L stock
    For 1 L media
    Trace elements*
    --
    1 ml
    NaCitrate.2H2O
    100 g
    5 ml
    FeCl3.6H2O
    10 g
    1 ml
    CaCl2.6H2O
    58 g
    1 ml
    MgSO4.7H2O
    100 g
    3 ml
    NH4NO3
    100 g
    3 ml
    KH2PO4
    100 g
    1 ml
    K2HPO4
    100 g
    1 ml
    *Trace Elements
    Component
    In 1 L stock
    H3BO3
    1.0 g
    ZnSO4.7H2O
    1.0 g
    MnSO4.H2O
    0.303 g
    CoCl2.6H2O
    0.2 g
    Na2MoO4.2H2O
    0.2 g
    CuSO4.5H2O
    0.063 g

References

  1. Chankova, S. G., Yurina, N. P., Dimova, E. G., Ermohina, O. V., Oleskina, Y. P., Dimitrova, M. T. and Bryant, P. E. (2009). Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii. J Ther Biol 34(7): 332-336.
  2. Chankova, S., Mitrovska, Z., Miteva, D., Oleskina, Y. P. and Yurina, N. P. (2013). Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats. Gene 516(1): 184-189.


How to cite: Chankova, S., Mitrovska, Z. and Yurina, N. (2013). Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells. Bio-protocol 3(15): e849. DOI: 10.21769/BioProtoc.849; Full Text



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