I3: Density Gradient Separation of Antigen Activated T Cells from Lymph Nod

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Tony Rook
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I3: Density Gradient Separation of Antigen Activated T Cells from Lymph Nod

Please find the link for the following protocol:

Density Gradient Separation of Antigen Activated T Cells from Lymph Node

Link Reference:
http://www.uchsc.edu/misc/diabetes/derc/protocols/immunologyprotocols.pdf

Populations

Materials
BSS
FBS
0.4μ syringe filter
sterile 15 ml centrifuge tube
sterile 15 ml polystyrene centrifuge
DMEM/10% FBS/ 50u/ml IL-2
sterile plugged pasteur pipettes
5.0 ml pipettes
10 ml pipettes
75 cm flasks

Procedure
Before taking the cells out of the incubator:
1. Prepare 10.0 ml BSS/15% FBS by adding 1.5 ml FBS to 8.5 ml BSS and filtering through a 0.4μ syringe filter into a sterile 15 ml centrifuge tube;
2. Filter approx. 4.0 ml Lympholyte M into a 15 ml sterile centrifuge tube;
3. Coat a sterile 15 ml polystyrene centrifuge tube with FBS by filtering approx. 2.0 mls of FBS through a 0.4μ syringe filter and after replacing the cap rolling the tube to coat it evenly on the inside surface, taking care not to get FBS on the cap, aspirate
the FBS, spin briefly in the centrifuge (bring up to 1500 rpm and shut off), aspirate the FBS that collected at the bottom of the tube;
4. Place 3.0 ml of the filtered Lympholyte into the FBS coated tube;
5. Prepare approx. 200 ml of DMEM/10% FBS/ 50u/ml. IL-2 and filter sterilize;
6. Freshly filter or prepare 200 ml of DMEM/10% FBS.
After the above steps are completed:
7. Harvest the cells from 24 well plates into a sterile 15 ml centrifuge tube by using a sterile, plugged pasteur pipette, titurating the cell suspension to resuspend as many cells as possible;
8. Pellet the cells by centrifugation for 5 min. @ 1500 rpm, aspirate off the medium and resuspend in 5.0 ml sterile BSS/15% FBS using a 5.0 ml pipette, add the remaining 5.0 ml BSS/15% FBS and mix by pipetting up and down, taking care not to draw the cells suspension into the cotton plug at the top of the pipette;
9. Draw the cell suspension into a 10 ml pipette tilt the tube with the lympholyte cushion and the pipette to approximately 45 and touch the tip of the pipette to the side of the tube approximately 1 cm above the Lympholyte, carefully layer the cell suspension over the 3.0 ml cushion of Lympholyte taking care to disturb the
interface as little as possible, when all of the cell suspension has been added place the tube upright and centrifuge at 1500 rpm for 15 min. with the brake off;
10. When the centrifuge has stopped carefully remove the tube and transfer to the hood upright and place into a rack, you should see a distinct tight band of cells at the interface of the lympholyte with the BSS/FBS, aspirate off approximately 7 - 8 ml of the BSS/FBS taking care not to disturb the band of cells, collect the cells in a plugged pasteur pipette by first squeezing the bulb and placing the tip of the pipette to the level of the band of cells and slowly releasing the bulb while sweeping the
interface area, it does not matter if you draw up some of the Lympholyte;
11. Transfer the cells to a fresh 15 ml centrifuge tube and fill with DMEM/10% FBS pellet by centrifugation @ 1500 rpm for 5 min. and wash twice more;
12. Resuspend the pellet in 5.0 ml DMEM/10% FBS/ 50u/ml. IL-2 and add 2.5 ml to each of two 75 cm flasks and divide the remainder of the DMEM/10% FBS/ IL-2 between the two flasks, gas and seal and place in the incubator;

Check cells daily for growth and if necessary, add additional DMEM/10% FBS/IL-2 to flasks, generally 100 ml at a time, if a flask with 200 ml of medium needs to be fed, split it into two separate flasks and bring each to a total of 200 ml.