95.2 mL

47.6 mL

23.8 mL

7.6 mL

Total volume

120 mL

60 mL

30 mL

10 mL

"Recombinant IL-2 (rIL-2) (14 U/mL) is added to the flask to facilitate T-cell expansion.

"Recombinant IL-2 (rIL-2) (14 U/mL) is added to the flask to facilitate T-cell expansion.

5. Centrifuge the cells at 330g for 5 min. Resuspend the cell pellet in 20 mL of fresh CM and add to a 162-cm2 flask with CM and rIL-2 according to Table 1.

6. Culture the cells for 4 d at 37°C and 5% CO2 with the flasks lying flat.

7. On day 4 of subculture (day 8 since restimulation), remove and save the supernatant (SN) in 50-mL tubes. Rinse the flask with 5 mL HBSS and save with SN. Add 7 mL dissociation buffer to each 162-cm2 flask (4 mL dissociation buffer for 75-cm2 flasks) and incubate for 3 min at 37°C. Strike the flask hard with the heel of a gloved hand to bring cells off the flask bottom (cells tend to come off in a sheet).

8. Resuspend the cells with a pipet. Pour the reserved SN back into the flask, mix, and remove an aliquot for counting. Note the volume for calculation of cell yield.

3.5.2. Preparation of Expanded T-Cell Lines/Clones for Injection

1. Centrifuge cells in 50-mL tubes at 330g for 10 min.

2. Remove SN and resuspend cells with HBSS. Pool cells into a 50-mL tube and bring to a volume of 30-50 mL with HBSS.

3. Wash cells by centrifugation at 330g for 10 min. Repeat the washing and resuspend cells in HBSS at the appropriate concentration for injection (2 x 108 cells/mL for intraperitoneal injection of 107 cells in 50 ^L).

4. Keep the cells on ice. Inject the cells as soon as possible. Mix well (do not vortex) before filling syringe. When loading syringe, remove needle and pull up cells (pulling cells through the needle will shear the cells).

3.5.3. Preparation of Diabetic Spleen Cells for Adoptive Transfer

1. Single-cell suspensions of spleen cells from diabetic NOD mice are prepared in HBSS using a glass tissue homogenizer and adjusted to 2 x 108 cells/mL.

2. For adoptive transfer experiments, mononuclear cells are counted, and cell viability is determined using trypan blue, a hemocytometer, and phase contrast microscopy or via a Coulter counter (see Note 10).

3. For phenotypic analyses, RBCs are lysed in spleen cell preparations by incubation in ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA at pH 7.2) for 5 min at room temperature before determining mononuclear cell counts and viability.

3.5.6. Monitoring of Recipient Mice for Glucosuria and Hyperglycemia

Beginning 5-7 d after adoptive transfer, recipient NOD or NOD.^a^ mice should be monitored daily for elevated urine glucose. When mice exhibit glucosuria, blood or plasma glucose levels should be determined. There are many reliable methods for testing circulating glucose levels. Some are more laborious than others. Most laboratories use handheld glucometers such as those used by diabetic patients. We currently use the Precision QID glucometer (MediSense, Waltham, MA). Mice with blood glucose levels above 16.5 mM (300 mg/dL) are considered overtly diabetic.

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