TheraKan Assay troubleshooting
Problems with creating a closed system in TheraKan™ devices ?
1.Devices were not closed- inner and outer chambers were not out of frame.
2. Inner chamber not pushed all of the way down, leaving a gap at the bottom in between the inner and outer chambers.
1.Swivel the inner and outer chambers so that chamber openings are not aligned to each other.
2.Make sure that the inner chamber is flush to the bottom of the outer chamber.
Chambers are difficult to assemble or swivel back and forth?
No or not enough lubricant was used.
Lubricant is provided with the devices to help facilitate device assemble and chamber movement. We recommend at least 1 application of lubricant all around the gasket of the inner chamber.
Immune cells were not detected in the TheraKan devices by flow cytometry or microscopy?
1. Immune cells were not labeled sufficiently.
2. Cell extraction from 3D cultures was not efficient for flow cytometry.
3. Not enough mmune cells were plated.
4. Incubation time not long enough.
5. Not enough cells in the 3D cultures.
6. 3D cultures did not contain enough viable cells.
1. Make sure the antibody labeling was sufficient for flow cytometry or microscopy.
2. Count your cells after extraction from matrix. Processing samples for flow cytometry tends to result in cell loss. Increase the number of cells in the test chamber and 3D cultures if necessary.
3. Increase the number of cells in the test chamber.We recommend starting with 500,000 cells/4 ml volume.
4. Increase incubation time to allow more immune cells to migrate from test chambers into devices. We recommend a time course experiment, incorporating a 24 hour timepoint.
5. Increase the numbers of cells in 3D cultures. We recommend starting with 100,000/250 ul matrix. You may want to test varying numbers to cells to opimize your experiment.
6. Make sure your cells were viable before they were embedded in 3D cultures by count cells with trypan blue. Make sure there was enough medium in devices. We recommend at least 500 ul or more.
3D cell cultures in devices were not viable ?
1. Not enough media in the TheraKan devices.
2. No serum in media or incompatible media.
3. Matrix not compatiable with cells.
4. Cells are contaminated.
5. Cells are senescent or overpassaged.
6. When adherent cells were being lifted off plate for seeding of 3D culture, they became over trypsinized.
1.We recommend 500 ul or more media in devices.
2. After establishing 3D cell cultures in devices, we recommend incubating 3D cell cultures in serum containing media for 24 hours to allow cells to recover before starting experiments. Make sure the media you are using is compatible with your cell line.
3. Make sure the matrix you are using is biocompatible.
4. Check for mycoplasm, bacteria, fungal or yeast contamination. Signs of contamination include: visible signs of particles under the microscopy, cloudy media and abrupt changes in pH in phenol red containing media. Repeat experiment. Add antibiotics to medium to reduce chances of contamination. Check reagents and equipment.
5. Check passage number. Use earlier passage cells.
6.Reduce concentration of trypsin. Reduce duration of tryspinization. Use a different method for cell dettachment. There are other enzymatic approaches that are commercially available that too many to list here, but search "alternatives or replacement of trypsin."