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General Protocol for Freezing and Thawing Cells

Comments:
Cells should be free of contamination in the form of bacteria, yeast, or fungi.
Mycoplasma testing should be performed prior to freezing.
Freezing media depends on the cell line.
One vial must be saved for testing the success of the freeze.
SECTION I: Adherent Cells
Materials:
Phosphate Buffered Saline PBS!
"rypsin#$%"& solution
"issue Culture Media
Cold Freezing Media usually '() dimethylsulfo*ide, %MSO!
+abeled Cryovials ,- per '((.mm plate for!
'((.mm plate of confluent cells
Freeze Procedure:
Pre-freeze
'! Chec/ for bacterial, yeast, or fungal contamination under a microscope.
0! "est a sample for mycoplasma using 1ibco2s Myco"ect /it Cat. 3o. '4560.('6!.
Freeze
-! "rypsinize cells standard protocol!.
7! 8e.suspend cells in media, transfer to a sterile centrifuge tube, centrifuge at '(((
8PM and 7C for -.4 min.
4! 8emove supernatant 9ith sterile Pasteur pipette.
5! :uic/ly re.suspend pellet by adding ' ml freezing media per vial to be frozen.
6! &li;uot ' ml freezing media plus cells per vial, and place on ice.
<! Freeze overnight at .<(C.
=! "ransfer vials to li;uid 3
0
tan/ for indefinite storage.
Post-freeze
'(! 8emove a vial from li;uid 3
0
tan/ and follo9 the tha9 procedure belo9 to test
the success of the freeze.
Thaw Procedure:
'! >arm tissue culture media 9ithout selection antibiotics to -6C, and label '((.
mm tissue culture plate.
0! 8emove vial from li;uid 3
0
tan/ and hold in -6C 9ater bath until sides are
tha9ed but center remains frozen.
-! 1ently pour cells into the plate. %o not sha/e the vial.
7! &dd = ml 9arm media drop9ise to the partially frozen cells.
4! Place plate in incubator.
5! Change media as soon as cells are attached remove %MSO a.s.a.p.!.
SECTION II: Susension Cells
Materials:
For Freeze
64 cm
0
".flas/s of cells in late log phase , 7( m+#".flas/!
Cold freezing medium usually contains '() dimethylsulfo*ide, %MSO!
+abeled cryogenic vials ,4 per 7( m+ volume of cells in late log phase!
For "ha9
Cold tissue culture medium
04 cm
0
".flas/
Freeze Procedure:
Pre-freeze
'! Chec/ for bacterial, yeast, or fungal contamination under a microscope.
0! "est a sample for mycoplasma using 1ibco2s Myco"ect /it Cat. 3o. '4560.('6!.
-! >hen cells have reached late log phase, determine cell density using Coulter
counter. Calculate total number of cells in flas/, and determine amount of freeze
medium needed. Cells should be resuspended in freeze medium at 4,(((,((( to
0(,(((,((( cells#m+.!
Freeze
4) Centrifuge cells in 4( m+ Falcon tube at '(((g for '4 minutes.
5) >hile cells are spinning, ma/e freeze medium e.g., =() FBS, '() %MSO!.
+abel cryogenic vials 9ith date, cell type, and user2s initials.
5! Suction a9ay supernatant from centrifuged cells and add freeze medium.
"riturate cells until homogeneous.
6! :uic/ly ali;uot ' m+ of freeze stoc/ per cryogenic vial. Scre9 each vial closed.
<! Put vials into storage bo* and place bo*, insulated 9ith paper to9els, into
"upper9are
?
container. Put entire container into @0(AC freezer.
=! &fter - hours, transfer container to @<(AC freezer and store overnight.
'(! 3e*t day, put cells into appropriate rac/ in li;uid 3
0
tan/.
Post-freeze
''! 8emove a vial from li;uid 3
0
tan/ and follo9 the tha9 procedure belo9 to test
the success of the freeze.
Thaw Procedure:
'! Slo9ly remove appropriate tray rac/ from li;uid 3
0
tan/. 8emove long safety pin
and ta/e out one vial from appropriate tray.
0! Put tray bac/ in slot and put safety pin bac/ in place. 8eturn tray rac/ to li;uid
3
0
tan/ and cap tan/ again.
-! 8apidly tha9 vial in -6AC 9ater bath until only a small ice pellet remains. Spray
do9n vial 9ith ethanol, 9ipe, and place into hood.
7! Pipet contents of vial , ' m+! into "04 flas/.
4! Slo9ly add 7 m+ of cold culture medium, at a rate of about ' drop every '(
seconds, s9irling occasionally. &dd another 4 m+ of culture medium.
5! Place flas/ in appropriate incubator.
6! Since freeze medium contains dimethylsulfo*ide %MSO!, spin do9n cells after
5.'0 hours and resuspend in fresh, pre9armed medium in ne9 "04 flas/.

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