I aim to establish a migration assay with human glioma cells. After migration over 16 h the migratory cells on the membrane underside should be characterised immunocytochemically with an anti EphB4 antibody.
Can you provide recommendations for this experiment? Will it be possible to visualise the staining in bright field microscopy?
ThinCert™ cell culture inserts are well suited for immunocytochemical staining. In principal bright field microscopy as well as fluorescence microscopy may be applied to the insert membrane.
In bright field microscopy membrane pores may interfere with the light that goes through the membrane (e.g. light scattering may occur). Bright field microscopy is therefore limited and may yield less brilliant images as fluorescence microscopy. If available fluorescence microscopy should be given priority over bright field microscopy. Optimum results may be obtained with confocal microscopy.
The following steps may be suggested:
1. Seed cells on insert membrane and let them migrate over 16-24 h (see also application note
“A quantitative cell migration assay using ThinCert™ cell culture inserts”)
2. Migration over 16-24 h
3. Removal of non-migratory cells on insert membrane using a cotton swab
4. Fluorescence staining of cells in the insert (see also application note
“The reconstruction and immunocytochemical characterisation of polarised epithelia in ThinCert™ cell culture inserts”)
5. Removal of insert membrane from insert housing, mounting on microscopy slide
6. Fluorescence microscopy (if possible confocal analysis)