Adherent cell selections

Selection of phage antibody libraries by panning adherent cells 

Grow the cell line under normal culture conditions on chamber slides to approx. 

Block the cells in 100 jd. PBS containing 3% skimmed milk for 1-2 h at 4°C for unfixed cells, or room temperature for fixed cells. 

Gently wash the cells with three changes of PBS. 

Ehite the phage which have bound to the cells by adding 100 p,l of fteshly made 100 mM triethylamine and incubating at room temperature for 10 min. 

Grow the cell line under normal culture conditions on chamber shdes to approx. 80% confluence (approx. 1 x 10 to 1 X 10 cells per well). Cells can either be left unfixed or fixed with a variety of fixation procedures, for example treatment with 0.1% ghitaraldehyde fiir IS min at room temperature. If unfixed cells are used keep the cells at 4°C at all times, since membrane proteins may be internalized at hitler temperatures. 

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Cellular adhesion FFF combines the controllable hydrodynamic shear forces of FFF and the selective adhesion of AC. The hydrodynamic shear is used to detach selectively and evenly adhered cells or particles from the surface and allows an estimation of the differences in cell/surface adhesion forces. The channel for cellular adhesion FFF is constructed in the same way as that used for Gr-FFF but is smaller in size and with the modification that the accumulation wall consists of either bare or polymer-coated surfaces. After the cell suspension is filled into the channel allowing sufficient time for cell adhesion, the flow is applied and fractions are collected. Despite the collection of fractions, cellular adhesion FFF can also be used as a tool to study rapid kinetics of cell surface adhesion, a largely unexamined area [332]. 

Selectivity determinations require two matched wells of adherent cells or two aliquots of nonadherent cells. The adherent cells should be seeded onto wells 24-48 h before the experiment. For nonadherent cells, use 2 million cells/phage. 

One of the early sources of adult stem cells to be investigated was bone marrow. A subset of multipotent progenitor cells was found to reside in the bone marrow niche and could be induced to differentiate down specific mesenchymal lineages [94], The bone marrow aspirate is collected from the iliac crest, followed by selection for plastic-adherent cells. 

Yeo WS, Hodneland CD, Mrksich M (2001) Electroactive monolayer substrates that selectively release adherent cells. Chembiochem 2(7-8) 590-593... 

Selection of phage antibo hbraries by panning adherent cells 77... 

Proximity selection using a guide antibody on an adherent cell line 80 Proximity selection using a natural ligand on cells in suspension 82... 

Alternatively, a two-step strategy can be conducted in which clones stably transfeeted with the ST are first selected and then further transfected with the protein of interest as shown in Fig. 11. This process is using a universal glycoengineered eell line which can be further transfected with the gene(s) of interest and selected to deliver high producer clones. Both methods have been tested at Siamed Xpress and successfully adapted to adherent cells or cells in suspension. 

Physical methods are reversible reformations, in the form of physical adhesion between two polymers. Physical adhesion, or immerse precipitation, does not change the interface. Therefore, the two polymers can easily be disunited using selective solvents. Adhering cell protein as the biomimetic substance and coating a substrate in a melting process are some examples of physical modification. However, homogeneous thickness of the conductive polymer cannot be achieved with these methods, resulting in various conductivities in different locations of the polymer. 

Cell Adhesion and Detachment, Figure 3 Selective adhesion of T iymphocytes in a microfiuidic device as a function of shear stress. The device geometry, shown in blue, is such that the fluid shear stress decreases aiong the iength of the device. This device was coated with the anti-CD5 antibody, which adheres to certain T lymphocytes. A mixture of T iymphocytes (stained green), and B iymphocytes (stained red) was passed through the device and the adhered cells were essentially all T lymphocytes (97% purity)... 

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