Counting and Plating Myd88, S2, and Dv-1 Cells for pAc5 Transfection
I want to see if my Myd88 cells are transfectable in they way I have been using. It is my thought that these cells are closer to what innubila primaries would be like, and so could give me an idea on how possible transfecting those cells might be.
Plate layout:
| PLATE 8 | 1 | 2 | 3 | 4 | ||||
|---|---|---|---|---|---|---|---|---|
| A | S2 control | Myd88 control | Myd88 control | Dv-1 control | ||||
| B | S2 2020 1ul reagent | Myd88 1ul 2020 reagent | Myd88 3ul 2020 reagent | DV-1 1ul 2020 reagent | ||||
| C | S2 2020 1ul reagent | Myd88 1ul 2020 reagent | Myd88 3ul 2020 reagent | DV-1 1ul 2020 reagent | ||||
I want to use the same amount of cells I have used for all of my other transfections, 1.5*10^5 or 150,000 cells/mL plating density. I will need 5mL total for the 3 Dv-1 and S2 cells, and I will want 15mL of the Myd88 cells to fill the 6 wells and 2 new flasks.
Dv-1 cells
- Scrapped of leftover cells from P30 flask (used earlier that day and re-fed) and removed the liquid
- Centrifuged the tube for 3 min at 400rpm
- Removed the supernatant
- Resuspended the cell pellet in 5mL 10% FBS Schneider’s medium
- 20ul from the resuspention was added to a hemocytometer and counts from each section of the hemocytometer were taken:
| section | quadrant 1 | quadrant 2 | quadrant 3 | quadrant 4 | section average |
|---|---|---|---|---|---|
| 1 | 82 | 63 | 81 | 76 | 75 |
| 2 | 93 | 85 | 73 | 102 | 88 |
- Each section was averaged, and the average of the two sections was taken
- Total DV-1 average: 81.5
- The cells per mL is the average * 10^4
- 81.5 * 10^4 = 815,000 cells per mL
- (8.15*10^5 cells/mL)(5mL) = (150,000 cell/mL)(xmL)
- x = 27mL medium
- Because I only want 5mL, I divided the xmL and the cell volume 5mL by 5
- 5.43mL 10% FBS medium
- 1mL cell resuspention
- This was made, and 1.5mL Dv-1 cell suspension was put in wells A4, B4, and C4
S2 cells
- Tapped the S2 flask and removed the liquid into a 15mL tube
- Centrifuged the tube for 3 min at 400rpm
- Removed the supernatant
- Resuspended the cell pellet in 5mL 10% FBS Schneider’s medium
- 20ul from the resuspention was added to a hemocytometer and counts from each section of the hemocytometer were taken:
| section | quadrant 1 | quadrant 2 | quadrant 3 | quadrant 4 | section average |
|---|---|---|---|---|---|
| 1 | 256 | 291 | 273 | 268 | 272 |
| 2 | 226 | 232 | 242 | 246 | 236 |
- Each section was averaged, and the average of the two sections was taken
- Total S2 average: 254
- The cells per mL is the average * 10^4
- 254 * 10^4 = 2,540,000 cells per mL
- (2.54*10^6 cells/mL)(5mL) = (150,000 cell/mL)(xmL)
- x = 84mL medium
- Because I only want 5mL, I divided the xmL and the cell volume 5mL by 14
- 6mL 10% FBS medium
- 0.36mL cell resuspention
- This was made, and 1.5mL S2 cell suspension was put in wells A1, B1, and C1
Myd88 cells
- Scrapped cells from the P20 flask and removed the liquid
- Centrifuged the tube for 3 min at 400rpm
- Removed the supernatant
- Resuspended the cell pellet in 5mL 10% FBS Schneider’s medium
- 20ul from the resuspention was added to a hemocytometer and counts from each section of the hemocytometer were taken:
| section | quadrant 1 | quadrant 2 | quadrant 3 | quadrant 4 | section average |
|---|---|---|---|---|---|
| 1 | 184 | 214 | 147 | 213 | 189 |
| 2 | 176 | 168 | 123 | 250 | 179 |
- Each section was averaged, and the average of the two sections was taken
- Total Myd88 average: 184
- The cells per mL is the average * 10^4
- 184 * 10^4 = 1,840,000 cells per mL
- (1.84*10^6 cells/mL)(5mL) = (150,000 cell/mL)(xmL)
- x = 61mL medium
- Because I only want 15mL, I divided the xmL and the cell volume 5mL by 4
- 15.25mL 10% FBS medium
- 1.25mL cell resuspention
- This was made, and 1.5mL Dv-1 cell suspension was put in wells A2, B2, C2, A3, B3, and C3.
The plate was put in the incubator to grow overnight