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Blood, Vol. 105, Issue 1, 31-39, January 1, 2005
The role of CCL21 in recruitment of T-precursor cells to fetal thymi
Blood Liu et al.
105: 31
Supplemental materials for: Liu et al, Vol 105, Issue 1, 31-39
Methods
Time-lapse visualization of thymus attraction. Ten µ L freshly neutralized collagen solution (2.16 mg/mL type I-A collagen in RPMI1640-based culture medium) containing 1×105 FL cells was placed in a 30-mm plastic dish and solidified at 37°C for 5 minutes. An E14.5 fetal thymus lobe that had been treated with 1.35 mM 2-deoxyguanosine (dGuo; Sigma) for 5 days was positioned approximately 0.5 mm away from the cells. The culture was submerged in 2 mL 0.5× collagen medium. Where indicated, antibody was included in the gel. The dish was placed in 70% O2, 5% CO2, and 25% N2 under an Axiovert S-100 microscope equipped with a digital CCD camera.11 The culture was time-lapse monitored for 20 to 24 hours using Openlab software.11 The numbers of cells that moved out of the initial cell spot and reached the thymus lobes were counted within visualized fields of identical size (as shown in Figure 1B and the Supplemental Movies). In some experiments, fetal thymus lobes were removed from the collagen gel, rinsed, and further cultured under conventional organ culture conditions.12,13
Thymic stromal cells and their reaggregates. dGuo-treated thymus lobes from E14.5 B6 fetal mice were dispersed with trypsin in EDTA.13,14 A slurry of 5×105 cells was deposited on a filter membrane and cultured for 24 to 48 hours to form reaggregated lobes.13
Files in this Data Supplement:
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