Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates





	1.	Refeed cells 2-3 hours before use.



	2.	Check each well for confluence and number of clones.  

            Selectively passage clones which have less than 10 

            colonies per well to another multiwell plate.



	3.	Aspirate off all of the media and wash 2 times with PBS.



	4.	Using the multi-channel pipetter, add 50 ul of trypsin 

            to each of the wells.  Change pipette tips between wells!



	5.	Incubate @ 37o C for 10-15 minutes.



	6.	Add 50 ul of 2X Freezing Media (60% DMEM, 20% FCS, 20% DMSO) 

            to each well and break up the colonies by pipetting up-and-down 

            about 5 times.



	7.	If required, remove 25 ul of the cell suspension and 

            pipette directly into a new multiwell plate.  If the 

            cells are being grown up for Southern analysis, use a 

            pre-gelatinized plate containing 200 ul of fresh M15 media 

            per well.  Alternatively, if the clones are being screened 

            using PCR, use a 96-well plate containing PCR Lysis Buffer 

           (50 ul + Proteinase K), lysing the cells either individually 

           or in appropriately mixed pools.



	8.	Using the multi-channel pipetter, add 100 ul of 

            filter-sterilized (0.22 um) Light Paraffin Oil to each well.  

            This prevents degassing and evaporation during storage @ -70o C.



	9.	Replace the lid on the plate and secure by completely sealing 

            with tape around all the edges.  Place the plate in a 

            polystyrene cuvette box, cover with a polystyrene lid 

            that fits, secure with tape, and freeze @ -70o C (the 

            temperature optimally should drop approximately 1o C/minute).



	10.	For samples being screened by PCR, take the plate of 

            cells in PCR Lysis Buffer (prepared in step 7, above) and 

            place @ 55o C for 1 hour to overnight.  Adequate steps must 

            be taken to avoid evaporation (e.g., addition of oil to 

            cover each well and/or incubation in a humidified chamber, 

            such as a sealed plastic container with a wet sponge inside).  

            After incubation, the lysates may be stored @ -20o C.  

            However, prior to use for PCR, the Proteinase K must be 

            inactivated by transferring the lysates to labelled 0.5 ml 

            Eppendorf tubes and heating @ 94o C for 15 minutes 

           (Program #99 in the PCR machine).



	11.	To retrieve ES cell clones that have been frozen by this  

            method, take the 96-well plate from the -70o C freezer and 

            place directly into the 37o C incubator.  Allow all of the 

            wells to thaw completely (this may take 10-15 minutes for 

            the wells near the center of the plate), then remove the 

            clones from the wells and transfer to appropriately labelled 

            wells in 24-well feeder plates pre-equilibrated with 2 ml of 

            M15 media per well.  For maximum recovery of sample, it is 

            important to vigorously pipette the thawed cells to dislodge 

            them from the bottom of the plate (where they settle during 

            the freezing process).  Since the cells tend to accumulate 

            around the perimeter of the wells as a consequence of the 

            minimal freezing volume, rinse the wells with media to further 

            facilitate maximum recovery and transfer to the appropriate 

            wells in the 24-well feeder plates.  There is no need to remove 

            the DMSO and the paraffin oil until the cells have replated 

           (24 hours after passage).