Isolation and Activation of Murine Lymphocytes

All mice are bred and maintained under specific pathogen-free conditions and all mouse protocols are conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee.

1. Preparation of Buffers and Reagents

1. Prepare complete Roswell Park Memorial Institute (RPMI) medium (10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, penicillin (100 IU/ml)/streptomycin (100 µg/ml), 55 µM 2-mercaptoethanol).
2. Prepare 20x Balanced Salt Solution (BSS) Stock 1 and Stock 2, Separately.
   1. Prepare 20x BSS stock 1 (111 mM dextrose, 8.8 mM potassium phosphate, 26.7 mM sodium phosphate dibasic in 1 L sterile water) and add 40 ml 0.5% Phenol Red to 20x BSS stock 1 before final volume adjustment. Sterile filter using 0.2 µm filter before storing at 4 °C.
   2. Prepare 20x BSS stock 2 (25.8 mM calcium chloride dihydrate, 107 mM potassium chloride, 2.73 M sodium chloride, 19.6 mM magnesium chloride hexahydrate, 16.6 mM magnesium sulfate in 1 L sterile water). Sterile filter using 0.2 µm filter before storing at 4 °C.
   3. To prepare 1x BSS for experimental use, dilute 50 ml BSS stock 1 and 50 ml BSS stock 2, separately, in 400 ml sterile water each. Combine both diluted solutions, adjust to pH 7, and add 20 ml FBS (2%). Top up to 1 L using sterile water and sterile filter using 0.2 µm filter.
      NOTE: BSS stock solutions should be prepared separately because the mixing of concentrated BSS stocks directly could result in precipitation.
3. Red Blood Cell (RBC) Lysis Buffer
   1. To prepare RBC lysis buffer, mix 9 parts stock ammonium chloride (155 mM ammonium chloride in sterile water) to 1 part stock Tris-base (130 mM Tris(hydroxymethyl)aminomethane in sterile water, pH 7.65) before use.
      NOTE: Store sterile stock solutions of ammonium chloride and Tris-base at 4 °C. Prepare lysis buffer fresh to ensure efficient lysis of RBCs.

2. Generation of Lymphocyte Suspension from Spleen or Lymph Nodes

NOTE: It is important to prepare all reagents and equipment required for the experiment before mouse euthanasia and to generate single cell suspensions of lymphocytes as soon as possible to maintain high cell viabilities.

1. Euthanize experimental mouse by cervical dislocation or CO₂ asphyxiation.
   NOTE: From this step onwards, all experimental procedures should be performed aseptically.
2. Dip the entire mouse into 70% ethanol before making any incisions. Remove the spleen and lymph nodes aseptically⁸, and place them in separate 15 ml tubes containing 5 ml ice-cold RPMI/FBS (RPMI with 2% FBS) or BSS/FBS (from step 1.2.3).
   NOTE: Since BSS prevents efficient RBC lysis, use RPMI for the preparation of the splenic cell suspension and switch to BSS after RBC lysis.
3. To generate a single cell suspension from spleen or lymph nodes, place the organ(s) in between two pieces of sterile 100 µm cell strainer mesh in a petri dish containing 2 ml ice-cold RPMI/FBS or BSS/FBS. Using the plunger of a 1 ml syringe, mash the organ(s) until it has been torn into very fine parts.
4. Transfer the cell suspension to a 15 ml tube and wash the cell strainer mesh with ice-cold RPMI/FBS or BSS/FBS. Collect the remaining cell suspension, add it to the same 15-ml tube, and spin down at 453 x g for 5 min at 4 °C. Remove the supernatant.
   NOTE: Re-suspend the pelleted cells by flicking the tube with fingers before adding RBC lysis buffer or medium in the subsequent steps.
5. Prepare room temperature (RT) RBC lysis buffer during centrifugation of the cell suspension (see step 1.3.). After pelleting the cells and removing the supernatant, re-suspend the cells with 1 ml RBC lysis buffer for every 10⁸ cells. Incubate the lysis reaction at RT for 3-4 min.
6. Stop RBC lysis with 14 ml ice-cold BSS/FBS and spin down at 453 x g for 5 min at 4 °C.

3. Purification of B and T Cells

1. Purification of B Cells
   1. Count the cells using a hemocytometer. Re-suspend up to 10⁸ splenic cells in 300 µl BSS/FBS and add 50 µl anti-CD43 magnetic microbeads^9,10. To remove dead cells, add 30 µl Annexin V magnetic beads. Incubate the cell suspension in a 4 °C refrigerator for 30 min.
2. Purification of T Cells
   1. Count the cells using a hemocytometer. Re-suspend up to 10⁸ cells in non-T cell depletion antibody cocktail (biotinylated antibodies against CD19, B220, Gr-1, TCR-γδ, CD49b, CD11c, CD11b, Ter119 and CD4 or CD8 depending on the target cell population to be purified), diluted 1:200 in 200 µl BSS/FBS^4,5. Incubate the cell suspension in a 4 °C refrigerator for 15 min.
   2. After incubation, add 10 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 °C. Remove the supernatant and re-suspend the cells in 165 µl BSS/FBS with 30 µl streptavidin microbeads and 15 µl Annexin V magnetic beads. Incubate the cell suspension in a 4 °C refrigerator for 30 min.
      NOTE: To ensure even labeling with the magnetic microbeads, incubate the cells with microbeads for 15 min, then mix the cell suspension gently by tapping the 15 ml tube and incubate another 15 min during step 3.1.1. or 3.2.2.
3. Preparation of Separation Column for Cell Purification
   1. Prepare an unused separation column during microbead labeling of the cells (step 3.1.1. or 3.2.2.). Pre-warm BSS (without FBS) to RT and use 2 ml to wash and equilibrate the column aseptically. After equilibration with BSS, the washed column should not be allowed to dry out.
      NOTE: We use the LS column instead of the recommended LD column due to its re-usability (see step 3.4.).
   2. After labeling with the magnetic beads, add 14 ml BSS/FBS to wash the cells and spin down at 453 x g for 5 min at 4 °C. Remove the supernatant and re-suspend the cells in 1-3 ml RT BSS.
   3. Attach a sterile 21 G needle to the tip of the column to reduce the flow rate during the process of purification. Load the cell suspension onto the equilibrated column and collect the flow through containing the purified target cells.
      NOTE: Avoid introducing bubbles into the column while loading.
   4. Wash the column once with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Reload the column with the flow through once again. Collect the flow through after the second loading in the same 15 ml tube.
   5. Wash the column 3 times with 1 ml BSS/FBS and collect the flow through containing the purified target cells. Thereafter, add 5 ml BSS/FBS to the column and, with a plunger, flush the magnetically labeled cells out of the column into a new 15 ml tube.
   6. Check the purity of the cells collected by flow cytometry using antibodies that bind to surface antigens of purified B or T cells^4,5.
4. Re-using the Separation Column
   NOTE: The LS column can be reused up to 4 times without affecting purification efficiency.
   1. Wash the column 3 times with 5 ml phosphate buffered saline (PBS) and 3 times with 5 ml distilled water from the top using the plunger.
   2. Wash the column with 5 ml 70% ethanol and dry the column extensively using an air tap to prevent the buildup of rust in the column.
   3. To prepare a used LS column for a separate purification experiment, wash the column from the bottom up with 5 ml 70% ethanol using the syringe adaptor. Next, wash the column from the bottom up twice with 5 ml sterile PBS, followed by 5 ml PBS once from the top of the column. Add 2 ml RT BSS to equilibrate the column and then proceed to loading the column with labeled cells.

4. CFSE Labeling and Stimulation

NOTE: Purified cells can be subjected to a variety of in vitro and in vivo functional assays. Here, we use purified T cells to determine the T cell stimulation capability of APCs⁵.

1. Pre-warm labeling solution (0.1% FBS in PBS) to 37 °C prior to CFSE loading.
   NOTE: Using a low percentage of FBS in PBS reduces cell death during CFSE loading and minimizes cell loss during centrifugation. However, too much FBS can interfere with CFSE loading.
2. Wash purified cells twice with labeling solution, then re-suspended at 2 x 10⁷ cells/ml in pre-warmed labeling solution in a 15 ml tube.
3. Prepare 10 µM CFSE solution (1:500 dilution of 5 mM CFSE stock solution) in pre-warmed labeling solution. CFSE solution should be freshly prepared each time to achieve optimal labeling.
4. To load cells with CFSE, add 1 part cell suspension to 1 part 10 µM CFSE solution in a 15-ml tube and incubate in the dark for 10 min at 37 °C. A final concentration of 5 µM CFSE is used to label 1 x 10⁷ cells/ml.
5. Invert the tube every 2 min to ensure a homogenous mixture of cells during CFSE loading.
6. To stop the reaction, add several volumes of ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 °C. Upon successful CFSE loading, the cell pellet will appear yellowish.
7. Wash CFSE loaded cells one more time with ice-cold complete RPMI medium and spin down at 453 x g for 5 min at 4 °C before using for in vitro culturing or in vivo stimulation.

5. In Vitro Stimulation

1. Prepare a 2x stock solution of stimuli (2x stimuli stock solution) immediately before use so that 100 µl of 2x stimuli stock solution can be added to 100 µl of cells to a final volume of 200 µl per well in a 96-well plate.
2. If a plate coated with stimuli (IgM or CD40 for B cells or CD3 and CD28 for T cells) is required, dilute the stimuli in PBS and pre-coat the culture plate at 4 °C overnight. Alternatively, the culture plate can be coated at 37 °C for 1 hr on the day of the experiment. Wash the coated plate twice with PBS (Do not allow the plate to dry out at any time).

Table 1: Concentrations of stimuli used to stimulate lymphocytes in in vitro culture.

3. For CFSE-labeled B cells, re-suspend to 3 x 10⁶ cells/ml in complete RPMI medium and culture in triplicate with 3 x 10⁵ cells/well in 96-well flat bottom plates for 72 hr.
4. For CFSE-labeled T cells, re-suspend to 0.5-3 x 10⁶ cells/ml in complete RPMI medium and culture in triplicate with 0.5-3 x 10⁵ cells/well in 96-well round bottom plates for 48 or 72 hr.

6. In Vivo Stimulation

1. For in vivo stimulation, adoptively transfer 4 x 10⁶ CFSE-labeled T cells per mouse (intravenously (i.v.) in 200 µl PBS) into each MHC-matched recipient mouse.
   NOTE: In this protocol, CFSE-labeled T cells can be adoptively transferred using tail vein or retro-orbital injection as these cells will home to lymphoid organs such as the spleen and lymph nodes.
2. Challenge the recipient mice one day later with the antigen.
   NOTE: In this example, we use ovalbumin (OVA protein, 50 µg/mouse) as the antigen because OVA-specific, T cell receptor (TCR)-transgenic T cells were adoptively transferred into recipient mice. Prepare OVA protein in sterile PBS and inject 100 µl of OVA protein/PBS or PBS control, via subcutaneous injection (s.i.), into each recipient mouse⁵.
3. Harvest and generate single cell suspensions from lymphoid organs (lymph nodes and spleens) of recipient mice 3 days after immunization with OVA protein or PBS. Separate lymph nodes into proximal lymph nodes (pLN), which includes axillary, brachial and superficial cervical lymph nodes) and distal lymph nodes (dLN), which includes mesenteric, popliteal, inguinal, lumbar, and caudal lymph nodes. Stain cells using the appropriate FACS antibodies to check for T cell proliferation.
   NOTE: In this CFSE cell tracking experiment, CFSE-labeled T cells from PBS-injected control mice establish a baseline fluorescence for non-dividing cells. Cell divisions of proliferating, antigen-stimulated, CFSE-labeled cells are visualized by measuring fluorescence peaks^12,13. With the PBS control, the number of cell divisions of proliferating, CFSE-labeled T cells can be determined^12,13.
4. Analyze the CFSE-labeled cell proliferation data by comparing the number of cell divisions or peaks between samples (Figures 1 and 2).
   NOTE: For example, CFSE-labeled, OVA-specific T cells adoptively transferred into recipient mice receiving the OVA antigen will undergo active proliferation compared to the PBS-injected control mice^5,12. Furthermore, it is noteworthy to point out that there are many ways to analyze CFSE cell proliferation data, as demonstrated by Hawkins and colleagues¹⁴.