If you are new to the TheraKan technology, we offer some tips to get you started and to help you obtain reliable data.
1. To learn how about establish 3D cultures in devices, read the instructions or watch the video demo.
2. To easily facilitate assembly and dis-assembly of devices, we recommend using the inert, non-toxic lubricant included.
3. Decide what size test chamber you want to use. We recommend a test chamber size of 3.5 cm in diameter, the well size found in a 6 well plate. Smaller test chambers may need to be tested by the user.
4. Decide what matrix you want to use to establish 3D cultures in devices. Rat tail collagen and Matrigel are commonly used matrices. However, there is no limitation to the type of matrix that may be used to establish 3D cultures in devices.
5. Decide the duration of your experiment. For cell migration into devices, we recommend a starting endpoint of 24 hours. Shorten or lengthen your experiment as needed.
6. You can control the size of the device opening to modulate flow of molecules or cells. If you are not sure what size opening to use, we recommend starting your assay by opening the devices completely.
7. For assistance in experimental design, please refer to the protocols section, where you may download individual protocols such as measurement of immune response and cytokine analysis. As we develop new protocols, this section will be updated, so check back from time to time.
1. To obtain reliable data, we recommend assaying devices in triplicate for each experiment.
2. Make sure there is sufficient media in the test chamber, so that the chamber opening of the device is submerged in media.
3.Few immune cells migrate to 3D matrix alone established in the TheraKan device. This makes for a reliable negative control for specificity.
4. The easiest method for detecting immune cell migration into devices is through inverted fluorescence microscopy. You can label your immune cells with a fluorochrome by expressing a reporter, protein tag or chemical label. Another reliable approach is flow cytometry of your 3D cultures. To prepare cells for flow cytometry analysis, you will need to dissociate cells from the matrix. For example, for collagen gels, you may use collagenase.
5. You can increase the efficiency of your experiments and control for assay variability by obtaining multiple measurements within the same device, including immune cell migration, cytokine expression and cell proliferation/survival in 3D cultures.
6. To obtain publication quality data, you may also refer to our publications section.