Counting and Plating S2 Cells and Adding 3.1 Cells to Plate 8 For Transfection

I left the 3.1 and 3.0 DinnDiNV cells that I tried my best to de-clump and plated in the 12 well plate on 4/27 for over a week, and two columns (the 3.0 cells that were scraped) just had not grown very much. I thought that there had just been too few clumps of cells to start growing out properly in those wells.

Plate layout:

PLATE 8 1   2   3   4  
A S2   3.0 top fluid, strained   3.0 bottom fluid, strained   3.1 top fluid, strained  
                 
                 
B S2   3.0 top fluid, strained   3.0 bottom fluid, strained   3.1 top fluid, strained  
                 
                 
C S2   3.0 top fluid, strained   3.0 bottom fluid, strained   3.1 top fluid, strained  
                 
                 

Here are the photos of every well on the 8th of May:

A2 B2 C2 A3 B3 C3 A4 B4 C4

The 3.1 cell fluid wells have a lot more growth, so I decided to keep those wells the same. I had 3.1 top of filter cells from 4/27 that had been growing in a flask since then, that I decided to trypsinize and add to the wells that hadn’t been doing good.

Adding more DinnDiNV cells

  • Poured off the medium from the 4/27 3.1 flask
  • Added 3mL trypsin, rinsed, and poured off
  • Added 5mL trypsin and waited ~5 minutes until all the cells had come off the bottom of the flask
  • Took the fluid and put it into a 15mL tube
  • Centrifuged the tube for 2 minutes at 200rpm
  • Removed all the supernatant from the pellet of cells
  • Resuspended the cells in 6mL 20% FBS Schneider’s medium with 4% mushroom
  • Ran those 6mL through a 70um cell strainer
  • Resuspended the top of the filter captured cells in 5mL of 20% FBS Schneider’s medium with 4% mushroom and plated that in a new flask for passage 3 of those cells
  • The 6mL that had passed through the 70um filter was distributed throughout the center 6 wells on plate 8, columns 2 and 3

Counting and plating S2 cells

  • I decided to plate the S2 at a slightly lower number than I have done previously, 100,000 cells per mL instead of 150,000 cells per mL, because I am not able to count the DinnDiNV cells and I guess that there are less of them
  • Took an old flask of cells and removed 3mL from the fluid (many cells in the supernatant) and put it in a 15mL tube
  • Added 3mL 10% FBS Schneider’s medium to the tube and mixed
  • Took 20ul from the cell fluid in the tube and put it in a hemocytometer to count the number of cells:
section quadrant 1 quadrant 2 quadrant 3 quadrant 4 section average
1 128 118 102 107 114
2 117 103 91 102 103
  • Each section was averaged, and the average of the two sections was taken
    • Total S2 average: 109
  • The cells per mL is the average * 10^4
    • 109 * 10^4 = 1,090,000 cells per mL
  • (1.09*10^6 cells/mL)(5mL) = (100,000 cell/mL)(xmL)
    • x = 65.4mL medium
  • Because I only want 6mL, I divided the xmL and the cell volume 6mL by 10
    • 6.54mL 10% FBS medium
    • 0.6mL cell resuspention
  • This was made, and 1.5mL S2 cell suspension was put in wells A1, B1, and, C1

The plate was put in the incubator to grow overnight