T and B cells from both donors in the recipients of 2 different donors respond normally upon the challenge of mitogens
| Responding cells . | n . | BrdU+ cells as % of T cells . | BrdU+ cells as % of B cells . | ||
|---|---|---|---|---|---|
| C57BL/6-derived . | SJL/J-derived . | C57BL/6-derived . | SJL/J-derived . | ||
| C57BL/6 → BALB/c | 5 | 8.48 ± 5.67 | NA | 4.94 ± 0.68 | NA |
| SJL/J → BALB/c | 5 | NA | 18.18 ± 3.80 | NA | 8.68 ± 1.51 |
| C57BL/6 + SJL/J → BALB/c | 9 | 12.93 ± 6.06 | 8.13 ± 5.57 | 3.90 ± 0.57 | 10.20 ± 2.86 |
| Normal mice | 5 | 7.23 ± 2.51 | 8.97 ± 2.24 | 1.93 ± 0.31 | 7.97 ± 0.86 |
| Responding cells . | n . | BrdU+ cells as % of T cells . | BrdU+ cells as % of B cells . | ||
|---|---|---|---|---|---|
| C57BL/6-derived . | SJL/J-derived . | C57BL/6-derived . | SJL/J-derived . | ||
| C57BL/6 → BALB/c | 5 | 8.48 ± 5.67 | NA | 4.94 ± 0.68 | NA |
| SJL/J → BALB/c | 5 | NA | 18.18 ± 3.80 | NA | 8.68 ± 1.51 |
| C57BL/6 + SJL/J → BALB/c | 9 | 12.93 ± 6.06 | 8.13 ± 5.57 | 3.90 ± 0.57 | 10.20 ± 2.86 |
| Normal mice | 5 | 7.23 ± 2.51 | 8.97 ± 2.24 | 1.93 ± 0.31 | 7.97 ± 0.86 |
More than 100 days after transplantation, 1.25 × 106 spleen cells per well were cultured with either immobilized anti-CD3 or anti-immunoglobulin-M in a flat-bottom 48-well culture plate at 37°C and 5% CO2 for 48 hours. A final concentration of 30 μM bromodeoxyuridine (BrdU) was added 24 hours before harvest. After culture, the cells were transferred into 6-mL tubes and washed once. The cells were then resuspended in 0.5 mL FACS Permeabilizing Solution for fixation and permeabilization. After 3 hours' incubation at 4°C, cells were washed twice and stained with anti-BrdU antibody (1 μg/mL) and anti-surface marker antibodies in the presence of DNAse I (Sigma) at a final concentration of 4 mg/mL for 30 minutes at room temperature. The stained cells were analyzed by means of a Coulter EPICS XL flow cytometer equipped with System II software. The values represent the mean ± SD. At least 10 000 T or B cells were analyzed for each sample.
BrdU indicates bromodeoxyuridine.
Not significant: C57BL/6 + SJL/J → BALB/c versus normal mice or single-donor recipients.