Marek: Freeze-down/Thaw

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==Materials==
==Materials==
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List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.
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List reagents, supplies and equipment necessary to perform the protocol here.
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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*10-50 ml centrifuge tube
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*reagent 1
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*cryo tubes
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*X μL reagent 2
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*DMSO, Tissue culture (TC)-tested (e.g. [http://www.sigmaaldrich.com Sigma], D2650)
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**component A (reagent 2 is made up of multiple components)
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*Cell culture medium (depends on cell line/cells)
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**component B
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*Trypsin-EDTA (e.g. from [http://www.paa.com PAA], L11-004) - for adeherent cells
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*equipment 1
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*'''Freeze-down medium'''
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*equipment 2
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**6-10% DMSO
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**Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
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*Basic TC centriguge (0 - 2500 rpm)
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*Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )
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==Procedure==
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==Freeze-down (suspension cells)==
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#Step 1
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#Centrifuge at 80-100 x g for 4-5 min at RT.
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#Step 2
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#Suck out the medium.
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#*Step 2 has some additional information that goes with iti.e. Keep at 4°C.
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#Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
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#Step 3
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#Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
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##Step 3 has multiple sub-steps within it.
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  *Leave over-night at -80 gr.C.
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##Enumerate each of those.
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*You can store cells at -80 gr.C for up to 6 months.
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*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).
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==Freeze-down (adherent cells)==
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#Wash cells with PBS.
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#Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
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#Observe cells under microscope until these are rounded.
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#Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
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#Centrifuge at 1000-1200 rpm for 4-5 min at RT.
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#Suck out the medium.
 +
#Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
 +
#Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).
 +
 
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==Thawing==
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#Keep frozen cells on dry ice until ready for thawing.
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#Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
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#Spray tube with 70% ethanol for sterilization.
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#Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
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#Fill centrifugation tube with culture medium: at least 10 ml total volume.
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#Centrifuge for 5 min at 80-100 x g (RT).
 +
#Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
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#Optional: Exchange medium next day.
==Notes==
==Notes==
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Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
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==References==
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'''Relevant papers and books'''
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<!-- If this protocol has papers or books associated with it, list those references here.  See the [[OpenWetWare:Biblio]] page for more information. -->
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<biblio>
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#Goldbeter-PNAS-1981 pmid=6947258
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#Jacob-JMB-1961 pmid=13718526
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#Ptashne-Genetic-Switch isbn=0879697164
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</biblio>
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==Contact==
==Contact==
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<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
<!-- You can tag this protocol with various categories.  See the [[Categories]] page for more information. -->
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[[Category:Mammalian cell culture]]
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[[Category:In vitro]]
[[Category:Protocol]]
[[Category:Protocol]]
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[[Category:Mammalian cell culture]]
 
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<!-- Move the relevant categories above this line to tag your protocol with the label
 
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[[Category:In vitro]]
 
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[[Category:In vivo]]
 
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[[Category:In silico]]
 
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[[Category:DNA]]
 
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[[Category:RNA]]
 
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[[Category:Protein]]
 
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Current revision

Contents

Overview

This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).

Materials

List reagents, supplies and equipment necessary to perform the protocol here.

  • 10-50 ml centrifuge tube
  • cryo tubes
  • DMSO, Tissue culture (TC)-tested (e.g. Sigma, D2650)
  • Cell culture medium (depends on cell line/cells)
  • Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
  • Freeze-down medium
    • 6-10% DMSO
    • Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
  • Basic TC centriguge (0 - 2500 rpm)
  • Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )

Freeze-down (suspension cells)

  1. Centrifuge at 80-100 x g for 4-5 min at RT.
  2. Suck out the medium.
  3. Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
  4. Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
*Leave over-night at -80 gr.C.
*You can store cells at -80 gr.C for up to 6 months.
*Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).

Freeze-down (adherent cells)

  1. Wash cells with PBS.
  2. Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
  3. Observe cells under microscope until these are rounded.
  4. Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
  5. Centrifuge at 1000-1200 rpm for 4-5 min at RT.
  6. Suck out the medium.
  7. Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
  8. Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).

Thawing

  1. Keep frozen cells on dry ice until ready for thawing.
  2. Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
  3. Spray tube with 70% ethanol for sterilization.
  4. Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
  5. Fill centrifugation tube with culture medium: at least 10 ml total volume.
  6. Centrifuge for 5 min at 80-100 x g (RT).
  7. Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
  8. Optional: Exchange medium next day.

Notes

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.


Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.

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