Collagen gel chamber assembly

Collagen mix was made by modifying the approach of Gunzer et al. (2000) using 5 ul 7.5%

sodium bicarbonate, 10 ul 10x MEM, 75 ml 3 mg/ml Bovine Collagen I (PureCol) and a chemokine, 1

ug/ml of CCL21 (300-35-100, Peprotech) or CCL19 (300-29B, Peprotech) or 0.3 ug/ml of SDF1-a/

CXCL12 (300-28A, Peprotech). All the work with collagen was done on ice to prevent polymerisa-

tion. 

Cell containing media (15 ul) supplemented with 10% Human serum (S-101B-FR, APS) (or 10%

FBS) is added to 35 ul of the collagen mix from before so that the final culture contained 200,000 CD8, 200,000

CD4s T-cells and DC at varying ratios (typically 1:1, 1:5 and 1:10 to T-cells). 

30 ul of the collagen is then added to a μ-slide angiogenesis chamber or sticky-Slide VI0.4 cham-

ber (ibidi) and put upside down in a 37°C incubator with 5% CO2 for ~60 min (to entrap the cells in

the gel as it polymerises). The wells are then topped up with 30 ml media containing the same con-

centration of chemokines as the gel (for m-slide angiogenesis) or 100 ml per well (for sticky-Slide

VI0.4).

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