Lymphocyte Isolation Protocol

Turn off UV light, turn on blower, leave outlets on.

Clean hood with ethanol

Discard all waste in biohazard containers-put sharps in small container on the floor

  1. Invert blood tubes a few times to gently mix. Pour blood into a 15 ml Falcon.NOTE THE TOTAL VOLUME OF BLOOD FOR EACH PERSON!! Spin at 1500rpm for 10 min. at room temp in the tissue culture centrifuge.
  2. You will end up with two layers--light layer=top, dark layer=bottom. If not two layers, spin for another 10 minutes. Take all of the top layer-it is ok to get some of the bottom layer as well.

2.Aliquot 5 mls Ficoll-Paque into a separate 15 ml Falcon –this will be done for you.

3.Pipette top lighter layer of spun blood onto the Ficoll-VERY GENTLY!!!

  • Don’t pour, don’t let blood go into the Ficoll
  • Empty the bottom layer into the blood waste bucket in lab hood, put used falcons into biohazard waste.

4.Spin 20 minutes at 2500-rpm room temp.

5.Transfer buffy coat to the tube containing 5 mls of PBS. Bring up the volume of this tube to 10 mls with PBS. Invert gently to mix. Take out 50 ul from each of your 4 tubes and put into 1 eppendorf tube to count-total volume is now 200ul.

6.Spin the PBS tubes at 1000 rpm for 10 min. at room temp.

7.Pour off the supernatant until you have about 400 ul in addition to your pellet.

8.For three of the four tubes for each person-resuspend your pellet in 1 ml Trizol and put in -80 freezer

9.Add 1 ml freezing media to the fourth pellet of each person and put into the cryovial. Immediately place cryovial into the blue/clear freezing container and store in - 80.

10.Ethanol down the hood-wipe dry with paper towels. Turn on the UV light, turn off blower, leave receptical on. Please leave the tissue culture room neat for the next person!

11.Count cells:

  1. Take hemocytometer out of ethanol and dry, also dry the cover slip-use kimwipes.
  2. Add ~15 ul to both sides of hemocytometer. Add cover slip.
  3. Using the 25 small square counting area in the middle-count the top two rows of 5 squares=10 squares total. Do this for both sides of the hemocytometer. Make note of both of these numbers.
  4. You do not have to do the calculations but for your information here is what to do: Average # cells x 2.5 x 10,000 x 10=number of cells per blood tube.