Assay for Quantitation of Cell-Cell Adhesion Using Fluorescent Dyes

Imhof, Beat A.; Schlienger, Claude; Handloser, Klaus; Hesse, B; Gisler, Roland H.

doi:10.1016/b978-0-12-442704-4.50023-2

Cited by 2 publications

(6 citation statements)

References 12 publications

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“…After removing the medium, FITClabeled proT cells, peripheral T cells, or polymorphonuclear leukocytes were added (105 cells/2001c1/well) and incubated at 20°C for 2 h. Unbound cells were removed by washing three times with DPBS containing 1 % BSA ; the plates were "flicked" dry between washes . Fluorescence, corresponding to cells bound in DPBS, was measured by a Fluoroskan II reader flitertek, Elfab Oy, Finland) (Imhof et al ., 1990) . The binding index is defined as the ratio of the number of cells bound to endothelium in the presence of blocking antibody to the number of cells bound in control medium .…”

Section: Cell Adhesion Assaysmentioning

confidence: 99%

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EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

Imhof

Ruíz

Hesse

et al. 1991

The Journal of Cell Biology

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Abstract. The mouse progenitor T lymphocyte (proT) cell line FTF1 binds in vitro to thymus blood vessels, the thymic capsule, and liver from newborn mice. A mAb, EA1, raised against an embryonic mouse endothelial cell line, blocked adhesion . The antibody also interfered with proT cell adhesion to a thymus-derived mouse endothelial cell line; it had no effect on the adhesion of mature T lymphocytes and myeloid cells. The antigen recognized by EA1 is located on the vas-

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Section: Cell Adhesion Assaysmentioning

confidence: 99%

“…FITC (Sigma Chemical Co., St . Louis, MO) was prepared as a 25 .7 mM stock solution in DMSO (Fluka AG, Buchs, Switzerland) and processed as described (Imhof et al, 1990). Briefly, 100 Kl stock solution was added to with SP 2/0 supernatant (control) or with EA-1 hybridoma supernatant (EA1) and used in the binding assay.…”

mentioning

confidence: 99%

EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

Imhof

Ruíz

Hesse

et al. 1991

The Journal of Cell Biology

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“…FITC (Sigma, St. Louis, MO) was prepared as a 25 mM stock solution in DMSO (Fluka, Buchs, Switzerland) and coupled to cells as described previously [43] with the following modifications: 10 p1 stock solution was added to 1 ml B cell suspension containing 5 x 106-40 x lo6 cells in Earle's BSS with glucose but without phenol red (EBSS; Gibco BRL) and incubated at 37 "C for 15 min in the dark. Labeled cells were then centrifuged (850 X g) through a layer of 5 ml 10% BSA onto a cushion of 28% BSA for 30 min at room temperature.…”

Section: Coupling Of Fitc To Lymphoid Cellsmentioning

confidence: 99%

“…Unbound cells were then removed from the CS 1.3 stromal cells by washing three times with Dulbecco's PBS (DPBS) containing 1% BSA. The level of fluorescence contained in each well, which corresponded to the amount of bound cells,was measured by aTitertek Fluoroskan I1 reader [43]. The binding index was defined by the ratio between the number of bound cells in the presence of inhibitory antibodies and the number of bound cells in the control medium.…”

Section: Adhesion Assays 25 In Vivo Injection Of Antibodiesmentioning

confidence: 99%

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Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

Imhof¹,

Schlienger²,

Handloser³

et al. 1991

Eur J Immunol

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B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.

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Assay for Quantitation of Cell-Cell Adhesion Using Fluorescent Dyes

Cited by 2 publications

References 12 publications

EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

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