Testing Running Dinn Cells Through Needles to Separate Cell Clumps using 25, 26, and 27 Gauge Needles
I am trying using needles on the cells again because there are 3 smaller sizes of needles that I can use, and I want to see if I can get no clumps. The other needles I used did a good job of getting rid of the large clumps, but small ones remained. I did not look at these cells under the hemocytometer before plating them because last time that did not seem to be very informative. I tried to do 10 passes through the needle for one well and 15 passes through the needle for the second well of each gauge. This was hard because the needles were so small it took a long time to do each well, so the number of passes might not exactly be right.
I am using only 3.1 cells this time because I have a lot of them.
Plate layout:
| 1 | 2 | 3 | 4 | |
|---|---|---|---|---|
| A | 3.1 no shearing | 3.1 25 gauge needled 10 strokes | 3.1 25 gauge needle 15 strokes | X |
| B | 3.1 no shearing | 3.1 26 gauge needled 10 strokes | 3.1 26 gauge needle 15 strokes | X |
| C | X | 3.1 27 gauge needled 10 strokes | 3.1 27 gauge needle 15 strokes | X |
- Poured off medium from flask
- Added 5mL trypsin and swished
- Poured off trypsin
- Added another 5mL trypsin and set flask 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 11mL of 10% FBS 4 % mushroom Schneider’s medium
- Took 3mL 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, I did this twice (A1 and B1)
- Aliquoted 1mL of the rest of the fluid into 6 1.5mL tubes (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 or 15 times, and then 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
The wells are imaged about every day to monitor how the cells grow and the break up of the clumps. Images can be found here