Key Specifications Table
|Key Applications||Detection Methods|
|Description||QCM Chemotaxis Cell Migration Assay, 96-well (3 µm), fluorimetric|
|Overview||Also available: Cell Comb™ Scratch Assay! Get biochemical data from a scratch assay! Click Here
Cell migration is a fundamental function of normal cellular processes, including embryonic development, angiogenesis, wound healing, immune response, and inflammation (1-2). One such process, leukocyte extravasation, is crucial for appropriate and effective immune response (3). Neutrophils normally exist in a resting state as they circulate though the body. However, upon interaction with small molecules known as chemoattractants, they rapidly respond with endothelial adhesion followed by emigration from the vasculature and chemotaxis to the site of inflammation (4-5). These chemoattractant receptors activate heterotrimeric GTP-binding proteins (G proteins) that initiate numerous elaborate signal transduction cascades, culminating in neutrophil migration and activation. Once at the site of inflammation, neutrophils respond with phagocytosis, superoxide generation, and the release of degradative enzymes.
Microporous membrane inserts are widely used for cell migration and invasion assays. The most widely accepted of which is the Boyden Chamber assay. However, current methods of analysis are time-consuming and tedious, involving cotton swabbing of non-migrated cells on the topside of insert, manual staining and counting. Recently a fluorescence blocking membrane insert was introduced to address these issues; however, this approach requires labeling of the cells with Calcein-AM and extensive washing to remove free Calcein before cell migration. The effect of this treatment on cell behavior/migration remains questionable.
The Chemicon QCM™ 96-well 3 μM Migration Assay does not require cell labeling, scraping, washing or counting. The 96-well insert and homogenous fluorescence detection format allows for large-scale screening and quantitative comparison of multiple samples (6).
In the Chemicon QCM™ 3 μM 96-well Migration Assay, migratory cells on the bottom of the insert membrane are dissociated from the membrane when incubated with Cell Detachment Buffer. These cells are subsequently lysed and detected by the patented CyQuant GR dye (Molecular Probes) (7). This green-fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids (8).
Most migration assays utilize an 8 μm pore size, as this is appropriate for most cell types, e.g. epithelial and fibroblast cells. The Chemicon QCM™ 3 μM 96-well Migration Assay utilizes a 3 μm pore size, which is appropriate for leukocyte migration. The system may be adapted to study different types of cell migration, including haptotaxis, random migration, chemokinesis, and chemotaxis.
The Chemicon QCM™ 3 μM 96-well Migration Assay provides a quick and efficient system for quantitative determination of various factors on cell migration, including screening of pharmacological agents, evaluation of integrins or other adhesion receptors responsible for cell migration, or analysis of gene function in transfected cells.
In addition, Chemicon also provides QCM™ 8μM 96-well Chemotaxis Cell Migration (ECM510) and Invasion Assays (ECM555) (8 μm pore), 24-well insert Cell Migration and Invasion Assay Systems, CytoMatrix™ Cell Adhesion strips coated with ECM proteins, or anti-integrin antibodies, and QuantiMatrix™ ECM protein ELISA kits.
The Chemicon QCM™ 96-well 3μM Migration Assay is ideal for studying chemotaxis cell migration. The quantitative nature of this assay is especially useful for large scale screening of pharmacological agents. The 3 μm pore size of this assay's Boyden chambers is appropriate for studying leukocyte migration. Each kit provides sufficient materials for the evaluation of 96 samples.
The Chemicon QCM™ 3μM 96-well Migration Assay is intended for research use only; not for diagnostic applications.
|Materials Required but Not Delivered||1. Precision pipettes: sufficient for aliquoting cells.
2. Harvesting buffer: EDTA or trypsin cell detachment buffer. Suggested formulations include a) 2 mM EDTA/PBS, b) 0.05% trypsin in Hanks Balanced Salt Solution (HBSS) containing 25 mM HEPES, or other cell detachment formulations as optimized by individual investigators.
Note: Trypsin cell detachment buffer maybe required for difficult cell lines. Allow sufficient time for cell receptor recovery.
3. Tissue culture growth medium appropriate for subject cells, such as RPMI containing 10% FBS.
4. Chemoattractants (e.g. 10% FBS) or pharmacological agents for addition to culture medium, if screening is desired.
5. Quenching Medium: serum-free medium, such as RPMI, DMEM, or FBM (fibroblast basal media), containing 5% BSA.
Note: Quenching Medium must contain divalent cations (Mg2+, Ca2+) sufficient for quenching EDTA in the harvesting buffer.
6. Sterile PBS or HBSS to wash cells.
7. Distilled water.
8. Low speed centrifuge and tubes for cell harvesting.
9. CO2 incubator appropriate for subject cells.
10. Hemocytometer or other means of counting cells.
11. Trypan blue or equivalent viability stain.
12. Fluorescence plate reader.
13. Sterile cell culture hood.
|Safety Information according to GHS|
|Storage and Shipping Information|
|Material Size||1 plate|
|Material Package||96 wells|
QCM Chemotaxis Cell Migration Assay, 96-well (3 µm), fluorimetric SDS
|Reference overview||Pub Med ID|
|Campylobacter jejuni invade chicken LMH cells inefficiently and stimulate differential expression of the chicken CXCLi1 and CXCLi2 cytokines.|
Charles L Larson, Devendra H Shah, A Singh Dhillon, Douglas R Call, Soohyoun Ahn, Gary J Haldorson, Chris Davitt, Michael E Konkel
Microbiology (Reading, England) 154 3835-47 2008
Campylobacter jejuni is a major food-borne bacterial pathogen, which is capable of causing diarrhoea containing blood and leukocytes. C. jejuni invasion of the intestinal epithelial cells and the release of proinflammatory molecules contribute to the pathophysiology of campylobacteriosis. Given the commensal relationship of C. jejuni with chickens, we hypothesized that C. jejuni invasion of chicken cells and the release of host cell cytokines would be significantly less than with human cells. To test our hypothesis, we examined the interactions of C. jejuni with chicken LMH cells, and performed in vivo experiments with chickens. The binding and internalization assays revealed that C. jejuni was significantly less invasive of LMH cells relative to human INT 407 cells, even though the bacteria bound to each host cell species equally. We also assessed interleukin-8 (IL-8) transcript, IL-8 secretion, and the release of chemoattractant molecules from the inoculated cells. Inoculation of LMH cells with C. jejuni stimulated expression of both chicken IL-8 orthologues, chCXCLi2 and chCXCLi1, but at levels significantly less than human IL-8 (huCXCL8) expressed from human INT 407 cells inoculated with C. jejuni. Moreover, the supernatant fluids of the C. jejuni-inoculated LMH cells resulted in little heterophil migration. In vivo, C. jejuni were observed bound to the cells lining the glandular crypts, but overt signs of cell invasion or pathology were not observed. These results indicate that cytokine expression in chicken LMH cells in response to C. jejuni is distinct from that of Salmonella typhimurium.