Study of SHIP‐binding cell surface proteins suggests c‐kit as a SHIP‐interacting receptor in mast cells

Ott, Vanessa; Moffitt, Lisa A.; Cambier, John C.

doi:10.1002/sita.200400029

Cited by 3 publications

(2 citation statements)

References 53 publications

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“…Unwanted cells bind to the beads which are retained by the magnet, and target cells are collected. Antibody to CD117 ( aka c-kit receptor) was used to capture the non-target RBL cells 35. Target cells from both the 10 3 and 10 6 samples were collected with yield, purity and viability as shown in Table I.…”

Section: Resultsmentioning

confidence: 99%

Microcup Arrays for the Efficient Isolation and Cloning of Cells

Sims

Allbritton

2010

Anal. Chem.

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Arrays of transparent, releasable micron-scale structures termed "microcups" were created for the purpose of patterning and isolating viable cells from small cell samples. Cells were captured by the microcups without the need for barriers or walls on the intervening substrate. Furthermore, in contrast to prior methods for creating cell arrays with releasable elements, no chemical modification of the substrate was required. Individual microcups were released from the array using a pulsed laser at very low energy. Improvements in microcup design enabled cells in suspension to be loaded into the microcups with greater than 90% efficiency. Cells cultured within the microcups displayed 100% viability and were cultured over 4 days yielding colonies that remained sequestered within the microcups to generate pure clonal populations. Standard microscopic imaging was used to identify cells or colonies of interest, and the microcups containing these cells were then released and collected. Individual target cells isolated in this manner remained viable as demonstrated by clonal expansion of 100% of collected cells. Direct comparisons with cell isolation by fluorescence-activated cell sorting and magnetic-bead-based isolation systems demonstrated that the microcup cell isolation procedure yielded higher purity, yield and viability than these standard technologies when separating samples with small numbers of cells. The power of this technique was demonstrated by the isolation of hematopoietic stem cells from a human bone marrow aspirate possessing only 4,000 total cells.Microfabricated arrays for patterning cells have been extensively described in the literature as they are ideal tools for performing fundamental studies of subcellular, intracellular and cell surface interactions. Applications range from patterning of neuronal networks, 1, 2 control of spindle formation, 3 stem cell studies, 4-9 gene expression monitoring, [10][11][12] design and manufacture of implant biomaterial, 13 drug screening, [14][15][16] and cell-based biosensors. 17,18 One general scheme for cell arraying employs modifying the array surface to create regions permissive to cell attachment and growth with non-permissive areas surrounding these regions. Such patterned surfaces can be created by microcontact printing, 19 photolithography, 20 injection bioprinting, 9, 21 optical tweezers,22 or magnetic forces.8 These surfaces have been used to create arrays of single or groups of cells, and have been used to control cell and colony morphology. This approach is used for adherent cell types that naturally grow attached to a surface.19 , 23 A drawback to these arrays is that under most circumstances individual cells displaying a unique attribute (cell surface marker, morphology, etc.) cannot be isolated for further study due to their attachment to the culture surface. Recently, Revzin's group successfully demonstrated the isolation of groups of cultured hepatocytes for genetic analysis from a micropatterned glass surface using a commercial laser mic...

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Section: Resultsmentioning

confidence: 99%

Microcup Arrays for the Efficient Isolation and Cloning of Cells

Sims

Allbritton

2010

Anal. Chem.

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“…Arrays with immobilized antibodies to c‐Kit and ErbB2 were used to enrich mixed populations containing RBL and SK‐BR‐3 cells, respectively. In these experiments, RBL cell lines are known to express the c‐Kit protein on their cell surface, while the SK‐BR‐3 cell line is a popular model system for the study of the ErbB2 receptor in breast cancer (47, 48). These cell surface proteins were chosen by virtue of their broad interest in clinical oncology and biological research, and their common use as cell surface markers for cell analysis and sorting (49–52).…”

Section: Resultsmentioning

confidence: 99%

Enrichment and expansion of cells using antibody‐coated micropallet arrays

2009

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Positive selection, sorting, and collection of single cells from within a heterogeneous population are required for many biological studies. We recently demonstrated a miniaturized cell array for this purpose; however, on-chip pre-enrichment and isolation of specific target cells would provide significant value for cell isolation. In the current work, mixed cell samples of fewer than 30,000 cells were used for panning by means of on-array antibody-capture to pre-enrich the target population. The cell surface receptors Fc e R 1 , c-Kit, and ErbB2 were used for positive selection of RBL, RBL, and SK-BR-3 cells, respectively, from the mixed population. The capture efficiency, selectivity, and enrichment for the target cells were calculated and compared with fibronectin-coated controls. As expected, the capture efficiency depended on the frequency of the target cell in the mixed population over the range of 0.3-33%. For a frequency of 5% target cells, the capture efficiency was 39%-53% for the three conditions, while the selectivity varied between 78% and 98% with 16-20-fold enrichment. Furthermore, single-cell cloning studies demonstrated a high cloning efficiency of target cells selectively isolated from the array. Antibody-based pre-enrichment in combination with micropallet-based cell selection will be a valuable tool for isolation and expansion of rare cells from small heterogeneous populations. '

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Phospholipid signalling: mediators in need of interdisciplinary techniques

Woscholski¹

2006

Signal Transduction

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Study of SHIP‐binding cell surface proteins suggests c‐kit as a SHIP‐interacting receptor in mast cells

Cited by 3 publications

References 53 publications

Microcup Arrays for the Efficient Isolation and Cloning of Cells

Microcup Arrays for the Efficient Isolation and Cloning of Cells

Enrichment and expansion of cells using antibody‐coated micropallet arrays

Phospholipid signalling: mediators in need of interdisciplinary techniques

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