Lymph Node Proliferation Assays and Establishment of Antigen Specific T Cell Lines
CFA (Freunds Complete Adjuvant)
0.14 M NaCl
2 x 1.0 ml Hamilton syringes
26 - 30 Ga. -inch needle
Antigens to be emulsified in CFA should be dissolved or dialyzed against 0.14 M NaCl and the pH adjusted to neutrality by either HCl of NaOH as required. Antigens are emulsified by drawing equal volumes of the antigen and CFA into 2 1.0 ml Hamilton syringes and mixing by pushing back and forth through a connecting needle. The antigen is properly emulsified when it becomes more viscous and when a drop placed on the surface of a beaker of cold water does not leave an oil slick. Immunization doses vary but typically range from 10 - 100 μg in 50μl CFA. Mice are immunized on the underside of the tail by
inserting a 26 - 30 Ga. 1/2 inch needle approximately 1/2 in from the base of the tail and insetting it full length.
Harvest of lymph nodes
The inguinal and peri-aortic lymph nodes are primed by this route and are harvested into a tube of sterile BSS. It is best to work quickly and sacrifice only 2 mice at a time. Once the
lymph nodes are collected they are disrupted by use of a ground glass homogenizer and processed as follows:
15 ml tubes
1. Spin at low speed to pellet debris by bring rotor up to 500 rpm and turning off the centrifuge
2. Transfer cell suspension to a fresh 15 ml tube, leaving pelleted debris behind
3. Spin cell suspension 1000 rpm 5 min. and wash by resuspension in 5 ml Clicks medium and pelleting @ 1000 rpm for 5 min. Repeat wash and resuspend in 5 ml Clicks
4. Take a 0.1 ml aliquot and dilute with 0.9 ml BSS and count on hemocytometer.
0.5% NMS (normal mouse serum)
flat bottom 96 well plates
saline (0.14M NaCl)
1.0 μCi 3H thymidine
1. Prepare Clicks with 0.5% NMS in advance (100 mls)
2. We generally use 1.0 X 106 cells per well in flat bottom 96 well plates in 0.2 ml Clicks and the larger cultures are set up with 1.0 X 107 cells in 2.0 mls, therefore either dilute or pellet and resuspend lymph node cells to 5.0 X 106 cells /ml.
3. The number of replicates is usually 2 - 4 depending on cell number and other desired use of cells.
4. Antigens must be sterile and are added in 10 μl saline, the dose varies with the purpose of the experiment and availability of antigen but typically ranges from 0.05 - 5.0 μM.
5. It is a good idea to perform serial dilutions of antigens, a convenient dilution factor is 1:3.16 (31.6 μl into 68.4 μl diluent) because every 2nd dilution gives 1:10 overall, we generally set up 6 dilutions of antigen.
6. Use only the inner 60 wells of a 96 well plate and add 0.2 ml BSS to the outer wells to prevent evaporation.
7. Plates are harvested from 3 - 6 days after the cultures are set up, but usually the plates are pulsed with 1.0 μCi 3H thymidine in the afternoon of day 4 and harvested
the next morning.
Larger cultures for the establishment of antigen specific lines
To obtain larger cell numbers for fusions and establishment of antigen specific T cell lines 1.0 X 107 cells are cultured in 2.0 ml Clicks. The appropriate concentration of antigen to be used should be established from the results of the proliferation assays. The cultures are incubated for 4 - 6 days, collected, pelleted and transferred into DMEM/10% FBS supplemented with 50 U/ml mouse IL-2 at a cell density of 2.5 - 5.0 X 104 /ml. These cells are culture for a further 8 - 9 days to give a total time of 14 days at which time they can be tested by proliferation assays and used to establish line as described.