Testing Running Dinn Cells Through Needles to Separate Cell Clumps
One of the main issues with the Dinn Cells is that they are very clumpy, to the point that they can be in clumps of 100s of cells and grow from there. Also when cells are trypsinized to release from the flask, the seem to be come even more clumpy. With clumpy cells, there is no way to count them and it might be bad for staining. I thought about trying to run the cells through needles to sort of shear up the clumps. I got three different needle gauges from the BioStore to try: 18, 20, and 23. The 18 is the largest opening and the 23 is the smallest that I tried. I planned to test this on both 3.1 and 3.0 cells
I put the cells in a 12 well plate, as well as looked at them in a hemocytometer (these images weren’t the best because of different amounts of cells in each sample)
| 1 | 2 | 3 | 4 | |
|---|---|---|---|---|
| A | 3.0 no shearing | X | 3.1 no shearing | X |
| B | 3.0 18 gauge needle | 3.0 20 gauge needle | 3.1 18 gauge needle | 3.1 20 gauge needle |
| C | 3.0 23 gauge needle | X | 3.1 23 gauge needle | X |
Each flask was done separately, but they were done exactly the same way, so this is written for both:
- Poured off medium from flasks
- Added 5mL trypsin and swished
- Poured off trypsin
- Added another 5mL trypsin and set flasks for 10 minutes, periodically gently tipping flask to remove cells
- Placed fluid in 15mL tube and centrifuged 3 minutes at 400rpm
- Aspirated off trypsin
- Resuspended cell pellet in 6mL of 10% FBS 4 % mushroom Schneider’s medium
- Took 2mL from this and plated it in a new flask to pass the cells, and added 5mL of fresh medium and set that flask aside
- Took 1mL from this and plated it in the 12-well dish in the no shearing well
- Took 20ul from the new flask (before adding the 5mL medium) and added that to the hemocytometer (tried to swirl beforehand) to image
- Aliquoted 1mL of the rest of the fluid into 3 1.5mL each (tried to keep amount of cells same between these but the last one got way more cells because of how fast they settle)
- For each 1.5mL tube with 1mL fluid, I ran it through a needle and a syringe 10 times (most of the volume each time), and then took 20ul and put in the hemocytometer to image, and the rest of the fluid went into the appropriate well in the 12-well plate
- The wells with cells had an extra mL of medium added to all of them
- The empty wells were given 2mL of fluid to help combat evaporation
Cells in the hemocytometer were not counted, just imaged. For many of these there were significant clumps making counting impossible, and uneven numbers of cells.
The plates and flasks were put in the incubator for the weekend.
The plate was imaged on 8/29, 4 days after plating. Shearing did seem to get rid of clumps, but not all of them. It is hard to tell if cells are happy after shearing. Because volumes are small and there were a lot of cells for some samples, it is hard to tell if cells are growing out, I think I will need to try smaller gauge needles and plating maybe in a flask or fewer well plate.
See images here