Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

1. Preparation

1. Before beginning, put on laboratory gloves and the appropriate protective clothing.
2. Sterilize all the dissection tools, first with a detergent and then with 70% ethanol and then dry thoroughly.
3. Prepare 50 mL of Hank's balanced salt solution (HBSS) containing 2% fetal calf serum (FCS).

2. Dissection

1. Using a carbon dioxide delivery system, euthanize the mouse by hypoxia. Secure the euthanized mouse on a dissection plate in the supine position and perform a longitudinal laparotomy using scissors and forceps.
2. Using forceps move the intestines and stomach on the right side of the abdomen to expose the stomach and spleen. The spleen is attached to the stomach.
3. Using forceps carefully detach the spleen from the stomach and place it on the Petri dish containing 5 mL of HBSS 2% FCS.

3. Immune Cell Isolation

1. Place the spleen on a 40 µm cell strainer over the Petri dish. Crush the spleen with a plunger to dissociate it.
2. Recover the adhered cells by rinsing the plunger and the strainer with 1 mL of HBSS 2% FCS.
3. Transfer the dissociated spleen and the fluid into a 15 mL centrifuge tube.
4. Wash the Petri dish with 5 milliliters of HBSS 2% FCS and transfer the fluid to the 15 mL tube.
5. Centrifuge the tube at 370 x g for 7 min at 10°C and discard the supernatant avoiding the pellet.
6. Resuspend the pellet in 2 mL of potassium ammonium chloride pipet up and down to resuspend the pellet and lyse the erythrocytes.
7. Wait for 2 min and add HBSS 2% FCS to the resuspended pellet to obtain the volume up to 14 mL.
8. Centrifuge the tube again at 370 x g for 7 min at 10°C. Discard the supernatant and resuspend the pellet in 5 mL of HBSS 2% FCS by pipetting up and down.
9. Count the cells using trypan blue staining assay and adjust the final cell concentration to 10⁷ cells/mL using appropriate volume of HBSS 2% FCS.

4. Adoptive Transfer

1. Transfer 2 mL of cell suspension obtained in a 5 mL collection tube.
2. Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatant.
3. Resuspend the pellet in 2 mL of PBS and centrifuge the tube at 370 x g for 7 min at 10°C.
4. Discard supernatant and resuspend pellet in 200 µL of PBS.
5. Using a 0.5 mL syringe with a 29G needle inject 200 µL of cell suspension into the experimental mouse intravenously into the retro-orbital blood sinus.
6. As a control, inject a second mouse in the same blood sinus with 200 µL of phosphate buffered saline.

5. Cells Harvest and Staining

1. Four days after the adoptive transfer, euthanize mice and remove the spleen.
2. Harvest the splenocytes as described in section 3.
3. Transfer 100 µL of cell suspensions from each mouse into two FACS tubes, labeled "control" and "transferred".
4. Centrifuge the tube at 370 x g for 7 min at 10°C and then discard the supernatants.
5. Prepare a mix containing the four antibodies at the dilutions listed in Table 1.

Antibodies	Fluorochrome	Dilution
CD45.1	BV711	1/200
CD45.2	APCCy7	1/400
CD4	BV786	1/1600
CD3	BV421	1/200

Table 1: Antibodies mix composition. Four antibodies cocktails preparation using concentrated antibody-fluorescent conjugates and HBSS.

6. Add 100 µL of the mix to each tube, and then incubate for 20 min on ice in the dark.
7. Add 1 mL of HBSS 2%FCS and then centrifuge the tubes at 370 x g for 3 min at 10°C.
8. Discard the supernatants and resuspend the pellets in 200 µL of HBSS 2% FCS.
9. Transfer the resuspended cells to new, labelled FACS tubes.
10. Using flow cytometry, as shown in FACS protocol, evaluate the presence of CD45.2 positive lymphocytes.

6. Data Analysis

1. Open the "FlowJo" software and drag the files for each tube in "All sample" window.
2. Double click on the "transferred" file to display the cells recorded from that sample on a dot plot that displays forward scatter "SSC-A" on the Y axis and side scatter "FSC-A" on the X axis.
3. Click on "polygon" and create a gating strategy to select lymphocytes, then distinguish donor and host cells using cell surface markers (CD45.1, CD45.2), and then characterize CD45.2⁺ cell population (CD3, CD4).
4. Repeat the analysis steps with "control mouse" file.
5. To visualize a cell population, click on "Layout editor".
6. Drag the "transferred cells" and the "transferred CD4 cells" population from "transferred" and "control" files into the "Layout Editor tab".
7. A dot plot representing CD45.2⁺ cells and CD4 lymphocytes will appear.
8. CD45.2 transferred cells should only appear in the "transferred mouse" dot plot.