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Caco-2/DC transwell system

MC Mariangela Cavarelli
SH Stéphane Hua
NH Naima Hantour
ST Sabine Tricot
NT Nicolas Tchitchek
CG Céline Gommet
HH Hakim Hocini
CC Catherine Chapon
ND Nathalie Dereuddre-Bosquet
RG Roger Le Grand
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Caco-2 cells (1 × 105) cultured in DMEM plus 10% FCS, were seeded on the upper face of a 6.5-mm filter (3-mm pore Transwell filter, Costar) in a 24-well plate for 7 to 8 days, until a transepithelial resistance (TER) ≥ 330 V/cm2 was achieved. DCs (4 × 105) were seeded on the opposite side of the filter for 4 h. Cell-free SIVmac251, a pool of 25% NSP or LSP, or a mixture of SIVmac251 and seminal plasma (ratio 1:1) were added to the apical side of the Caco-2 cells for 1.5 h. Negative controls consisted of medium (DMEM 10% FCS) or supernatant from a uninfected CEMx174 culture. Caco-2/DC filters were fixed in 2% paraformaldehyde (PFA) for successive evaluation by confocal microscopy. A list of the antibodies used for the confocal microscopy analysis of Caco-2 cells and DCs is provided in Supplementary Table 1.

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