Counting and Plating Myd88 and Dv-1 Cells to Infect Them With DiNV
The plan is to do full replication this time, 3 replicates for every treatment/condition, for both day 0 and day 5. I will do this in 12 well plates, so I will need only 2 plates. I want to seed cells at 100,000 cells per Myd88 well and 75,000 cells per Dv-1 well. This is more than I did previously but that should be fine hopefully.
Plate layouts:
| Day 0 | 1 | 2 | 3 | 4 | ||||
|---|---|---|---|---|---|---|---|---|
| A | Dv-1 Infected with DiNV replicate 1 | Myd88 Infected with DiNV replicate 1 | Dv-1 not infected replicate 1 | Myd88 not infected replicate 1 | ||||
| B | Dv-1 Infected with DiNV replicate 2 | Myd88 Infected with DiNV replicate 2 | Dv-1 not infected replicate 2 | Myd88 not infected replicate 2 | ||||
| C | Dv-1 Infected with DiNV replicate 3 | Myd88 Infected with DiNV replicate 3 | Dv-1 not infected replicate 3 | Myd88 not infected replicate 3 | ||||
| Day 5 | 1 | 2 | 3 | 4 | ||||
|---|---|---|---|---|---|---|---|---|
| A | Dv-1 Infected with DiNV replicate 1 | Myd88 Infected with DiNV replicate 1 | Dv-1 not infected replicate 1 | Myd88 not infected replicate 1 | ||||
| B | Dv-1 Infected with DiNV replicate 2 | Myd88 Infected with DiNV replicate 2 | Dv-1 not infected replicate 2 | Myd88 not infected replicate 2 | ||||
| C | Dv-1 Infected with DiNV replicate 3 | Myd88 Infected with DiNV replicate 3 | Dv-1 not infected replicate 3 | Myd88 not infected replicate 3 | ||||
Because there will be 12 wells for each cell type, and 1.5mL in each well, I will need ~18mL of properly diluted cells to separate out.
Dv-1 cells
- Tapped off cells from P30 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 | 176 | 172 | 168 | 186 | 175 |
| 2 | 185 | 202 | 177 | 165 | 182 |
- Each section was averaged, and the average of the two sections was taken
- Total DV-1 average: 178.5
- The cells per mL is the average * 10^4
- 178.5 * 10^4 = 1,785,000 cells per mL
- (1.785*10^6 cells/mL)(5mL) = (75,000 cell/mL)(xmL)
- x = 119mL medium
- Because I only want 18mL, I divided the xmL and the cell volume 5mL by 5
- 23.8mL 10% FBS medium
- 1mL cell resuspention
- This was made, and 1.5mL Dv-1 cell suspension was put in wells A1, B1, C1, A3, B3, and C3 in both plates
Myd88 cells
- Scraped cells from P20 flask and removed the liquid
- Centrifuged the tube for 3 min at 400rpm
- Removed the supernatant
- Resuspended the cell pellet in 3mL 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 | 212 | 215 | 196 | 256 | 219 |
| 2 | 201 | 204 | 170 | 191 | 191 |
- Each section was averaged, and the average of the two sections was taken
- Total Myd88 average: 205
- The cells per mL is the average * 10^4
- 205 * 10^4 = 2,050,000 cells per mL
- (2.05*10^6 cells/mL)(3mL) = (100,000 cell/mL)(xmL)
- x = 61.5mL medium
- Because I only want 18mL, I divided the xmL and the cell volume 5mL by 3
- 205mL 10% FBS medium
- 1mL cell resuspention
- This was made, and 1.5mL Dv-1 cell suspension was put in wells A2, B2, C2, A4, B4, and C4 in both plates
- Both plates were put in the incubator to wait for 2 days