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Mosaic expression of membrane peptidases by confluent cultures of Caco‐2 cells

“…When the primary antibody was replaced with a non-immune serum control, no immunofluorescent staining was observed. As previously reported, staining on intact cells was found to be homogeneous for DPPIV [4,7] and heterogeneous for E-24.1 1 [6,8]. Indirect immunofluorescent staining of ACE, AP-N and AP-W showed surface staining on some but not all of the cells.…”

“…Using indirect immunofluorescent staining, the membrane peptidase dipeptidylpeptidase IV (DPPIV) has previously been shown to be homogeneously distributed on the surface of Caco-2 cells 14.73. In contrast, we recently reported that another ceil-surface peptidase, endopeptidase-24.1 I (E-24.1 I), was present on the surface of some cells but not on others [6]. Jalal et al [8] have confirmed this observation and also shown that E-24.1 1 is in fact expressed by all cells but in some is confined to an intracellular pool, and aminopeptidase W (AP-W) were also detected only on the surface of some cells, but found to be located in intracellular pools in the majority of the cells examined.…”

“…In the present study, the cell-surface expression of five membrane peptidases on confluent Caco-2 cells has been investigated and evidence is presented to show that the mosaic pattern of E-24.11 surface staining is also observed for other cell-surface peptidases. Angiotensin converting enzyme (ACE), amino~ptidase N (AP-N) Caco-2 cells were grown to confluency on glass coverslips in six-well plates (Linbro, Flow Laboratories, Rickmansworth, UK) using Dulbecco's minimal essential medium supplemented with 20% beat-inactivated fetal calf serum (both Imperial Laboratories, Hounslow, Middlesex, UK), 1% non-essential amino acids, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all Gibco BRL, Uxbridge, Middlesex, UK) at 37°C in a 5% COJ95S air atmosphere with the medium changed 48 h after seeding and daily thereafter [6]. Cells were seeded at 1.2 x lo4 cells/cm' and used between pasages 93 and 98.…”

“…Following a 10 min incubation with 2 ml NH&l (SO mM in PBS), the coverslips were washed again and non-specific binding sites blocked by the addition of 2 ml of gelatin (0.04% w/v m PBS) for 15 min. After washing, coverslips were then placed on a 0.1 ml drop of primary antibody (RP161, RP181, RP125, RH179 or 22A5 diluted 1500 in PBS gelatin) for 1 h. Donkey anti-rabbit (or anti-mouse for 22A5) FITC-conjugated IgG secondary antibody (diluted 1:50 in PBS gelatin) was centrifuged (8000 x g) before use and 0.1 ml of the supernatant applied to washed coverslips for 30 mm at 4°C [6]. Following further washing, the coverslips were mounted on microscope slides and viewed either by epi-illumination using a Zeiss microscope or by Leica scanning confocal laser microscopy.…”

“…On reaching confluency Caco-2 cells undergo differentiation into enterocyte-like cells and express many hydrolases on the apical brush border [3--61. These hydrolases include some disaccharidases, for example sucrase-isomaltase and lactase-phlorizin hydrolase [4], and numerous peptidases [6]. Using indirect immunofluorescent staining, the membrane peptidase dipeptidylpeptidase IV (DPPIV) has previously been shown to be homogeneously distributed on the surface of Caco-2 cells 14.73.…”