Science Gateway

Protocols: ES Cell Culture - Gene Targeting - Protocols

1.	Preparation of Feeder Layers And SNL Stocks


1.1.	Gelatinizing Plates
Prepare plates by covering surface with 0.1% Gelatin solution:

	Plate Size (cm)	Amount of Gelatin (ml)
	3	2
	6	4
	10	9
	15	18

Swirl the plates to cover the entire surface.  Allow plates to gel 
@ room temp. for at least 2 hours.  Aspirate off all of the gelatin 
directly before using the plates.


1.2.	Feeder Layers

Use the 15 cm stock plates to make feeders.

	1.	Remove 12 mls of the media from each plate.

	2.	Add 360 ul Mitomycin C stock solution to each plate and 
            swirl to cover surface.

	3.	Incubate @ 37o C for 2 hours.

	4.	After incubation, aspirate off all of the media and wash 
            the cells twice with PBS, 5-10 ml per wash (enough to cover 
            the surface).  Aspirate off all of the PBS.

	5.	Add 2 ml of Trypsin to each 15 cm plate.  Swirl to cover 
            the entire surface.  Incubate @ 37o C for 5 minutes.

	6.	Add 5 ml of media (7% FCS, 1X GPS) to each plate to 
            inactivate the trypsin.  Suspend the cells by pipetting 
            up-and-down and transfer the suspension to sterile 15 ml 
            centrifuge tubes.  Repeat the suspension process, pooling 
            all of the suspensions into the 15 ml tubes, until all of 
            the cells have been harvested.  (You should be able to pool 
            suspensions from two 15 cm plates into one 15 ml tube.)

	7.	Spin the tubes for 7 minutes @ 1,000 rpm.

	8.	Carefully aspirate off the supernatant.  Do not touch the  
            cell pellet.  Tap each tube gently to break up the pellet.

	9.	Add 5 ml of media to each tube and resuspend the cell pellets.  
            Vigorously pipette up-and-down to insure a good 
            suspension.  Finally, pool all of the suspensions 
            into one 50 ml tube.  It is imperative that the 
            total volume of media added to resuspend the cells 
            be recorded; that value will be used later in calculations.

	10.	Transfer 200 ul of the cell suspension to a plastic vial 
            and add 10 ml PBS or Isoton II.   Count the cells using the 
            Coulter Counter.

		Calculating the Cell Number (only relevant if the 
            counter is set to count a 0.5 ml sample):

		The Coulter Counter will display "XXXXX", which
			= "X,XXX,X00 cells/ml"
			= "X.X x 106 cells/ml"

		To calculate the total number of cells, multiply 
            "X.X x 106 cells/ml" by the volume from which the 200 ul 
            aliquot was taken (see step 9).

	11.	We require 3.5 x 105 cells/ml.  Therefore, in order to 
            reach this cell density, you must calculate the volume 
            of media into which the cells will be added.  To do this, 
            divide the total cell number by 3.5 x 105 cells/ml.

		Example:

		After centrifuging my cells and aspirating off the media,  
            I have 3 cell pellets to resuspend.  Therefore, I use a total 
            of 15 ml of media to resuspend the 3 pellets.  I then count 
            a 200 ul aliquot of the suspension, and the Coulter Counter 
            displays 6 9 5 7 9

			= 6,957,900 cells/ml

			= (6.9 x 106 cells/ml)  x  15 ml (total 
                     volume used to resuspend cells)

			= (1.035 x 108 cells total)  /  (3.5 x 105 cells/ml)

			approx. = 296 ml media needed

	12.	Once you have determined the required volume of media, add 
            the cell suspension to that volume of media and shake 
            to uniformly distribute the cells.

	13.	Check to see if the proper cell density has been reached  
            by taking a 200 ul aliquot of the media and counting as 
            before.  However, when calculating the total cell number, 
            be sure to use the total volume of media to which the cells 
            were added.

		Example:

		From the previous example, we calculated that 296 ml of 
            media was needed to reach the required cell density.  Upon 
            recounting to check the density, the Coulter Counter 
            displayed 0 4 3 5 8

			= 435,800 cells/ml

			= (0.43 x 106 cells/ml)  x  296 ml (total volume to  
                     which cells were added)

			= (1.2728 x 108 cells total)  /  (3.5 x 105 cells/ml)

			approx. = 364 ml media needed

		If the total cell count is still too high to give the  
 desired density, add the determined amount of media that will dilute 
 the cells to the proper density (i.e., the difference between the 
 volume of media needed for the first count and the volume 
 needed for the second count).

		Example:

		From the second determination above, we found that 
            364 ml of media is needed to obtain the desired cell 
            density.  Since we have already added 296 ml of media, 
            we need only add another 68 ml.

			364 ml  -  296 ml = 68 ml of media to be added

	14.	Once the required cell density (3.5 x 105 cells/ml) has 
            been reached, aliquot the prepared Feeder Media into the 
            size and numbers of feeder plates needed (remembering to 
            use plates that have been pretreated with the gelatin).  
            Swirl the plates to uniformly distribute the cells.

	Size of Plate (cm)     Amount of Media to Aliquot (ml)
	 	15	                30
		10	                12
		6	                 4
		3 (or 6-well)          2
		24- or 4-well          0.5

	15.	Place the feeders in the 37o C incubator, labelling the tray 
            with "Feeders" and the date.


SNL (STO-Neo-LIF) Stocks


Use a confluent 10 cm plate to prepare stock plates.  Gel 15 cm 
plates for the stocks and one 10 cm plate for the passage plate.

	1.	Aspirate off all of the media and wash the cells twice with 
            PBS, 4-7 ml per wash (enough to cover the surface).  Aspirate 
            off all of the PBS.

	2.	Add 0.8 ml Trypsin to the plate; swirl to cover the 
            entire surface.  Incubate @ 37o C for 5 minutes.



	3.	Add 10 ml of media to the plate to inactivate the trypsin.  
            Vigorously pipette up-and-down to suspend the cells.

	4.	Determine the total volume of media that will be needed for 
            the stock plates based on the following:

		Size of Plate (cm)     Amount of Media to Aliquot (ml)
			15	                     30
			10	                     12

	5.	Add the cell suspension to the required volume of media.  
            Shake the bottle to insure a uniform distribution of cells.

	6.	Aliquot the STO-Neo Media into plates in the volume specified 
            for each size of plate (see step 4).  Remember to use plates 
            that have been pretreated with gelatin.

	7.	Place the stock plates in the 37o C incubator, labelling 
            the tray with the clone number (e.g., SNL 76/7), the passage 
            number, and the date.


NOTE:	The 15 cm plates will be used upon confluence to prepare the feeder 
plates.  The one 10 cm plate will be used to maintain the cell line through 
a number of passages.