Applications and FAQs
Basic Protocol 1
- Seed and grow cells on chamber slides
- Remove medium
- Wash cells 1x with PBS or CellCover
- Place slide in CellCover and store at 4°C until use
- Proceed to staining protocol according to experimental design, e.g. immunostaining (If RNA is to be isolated for downstream application, you can stain cells by using CellCover)
Basic Protocol 2
Basic immuno-staining protocol for suspension cells:
- Harvest cells by centrifugation
- Remove supernatant
- Flick tube to suspend cells in residual medium
- Add CellCover (5 to 10x volumes) and store at 4°C until use
- Pellet cells and remove SN
(Some cells might need higher centrifugation speed for pelleting.)
- Proceed to standard staining protocols, e.g. immunostaining (If RNA is to be isolated for downstream application, you can stain cells by using CellCover as antibody diluent and washing buffer
Basic Protocol 3
Basic protocol for tissue specimens:
- Cut tissue into approximately 5 mm pieces
- CellCover does not penetrate tight junctions, thus encapsulated organs must be open across the largest diameter.
- Place tissue in CellCover (at least 10x volumes) and store at 4°C until use
- Change CellCover at least once after 4-24 hours
- Proceed according to experimental design e.g. RNA isolation
Material Safety Datasheet
Can I store my purified RNA in CellCover?
Yes, you can. Storage of RNA in CellCoverprotects your RNA sufficiently for most subsequent purposes.
How shall I store my purified RNA in CellCover?
This really depends on when do you need it. After purification you usually have it on ice. If you need it for e. g. a Northern analysis within the next two days, keep it on ice (best in a cold room for the ice lasts longer). If you are not sure when you start your subsequent analysis, freeze it at minus 20°C. However, if you want proper maintenance for several month consider storage at -80°C.
My purified RNA is partly degraded, what might have been wrong?
Degradation, usually from the 5´end, is a common problem when using purification kits. We recommend to strictly adhere to instructions of the manufacturer of the kit and to work as cold as possible (above 0°C). After precipitation, we recommend dissolving purified RNA in ice-cold CellCover.
Does my purified RNA degrade in CellCover?
RNA protection by CellCoveris almost as effective as deep freezing and sufficient for most purposes (e.g. no change in RIN after 24 h on ice).
What are typical RIN values if using CellCover?
RIN values using CellCover are generally among the best you can get. You can expect RIN values of 10 even after storing cells for days in a fridge. However, many variables influence the individual experiment, e.g. RIN value increases the more experienced the researcher is, is dependent on the kit used for purification and might differ from cell type to cell type.
In tissue, how long does it take until my RNA is protected with CellCover?
CellCover works on to ends, chemical stabilisation and protection against enzymatic degradation. Time to fully protect RNA increases in the following order: non-adherent cells- adherent cells – spheroids – tissue. As a guideline: the more cells, the longer it takes. Usually storing a 5 mm tissue block sample on ice o/n is sufficient. Store spleen or central nervous system tissue for at least 24 h on ice.
Can I freeze my tissue samples in CellCover?
You can freeze your sample in CellCover, if protein in your sample is of interest for you. However, you should leave your sample on ice o/n for some time before freezing.
Is there something important to know when isolating RNA from tissue in CellCover?
Any manipulation might affect your RNA. Cell lysis is one of the most critical steps for it must be fast and efficient. Cells can be resuspended in a small volume CellCover before adding Lysis Buffer. Tissue should be minced on ice or grind under liquid nitrogen before adding Lysis Buffer.
Can I analyse my cells in CellCover with MALDI?
Yes, you can. Follow your lab protocol. Ingredients of CellCover do not interfere with macromolecular analyses.
Does CellCover protect HMW-RNA?
CellCover protects5´prime ends of RNA , so you can analyse large RNA species more easily. Some 20 kbases should not be a problem.