I am learning about seeding wells and I am honestly confused with the whole process, such as the calculations, what is a surface area of a 6 well or 24 well plate and how am I supposed to use the information below for this process? Can someone explain what this information means below and how I can use this information when seeding cells in lab? Thank you.
3T3 18hr doubling time
Final count on confluent plate
2 days ahead
# cells to seed
3 days ahead
# cells to seed
4 days ahead
# cells to seed
3cm or 6 well
1 x 106
2.5 x 105
1 x 105
2.5 x 104
6cm or T-25
2 x 106
5 x 105
2 x 105
5 x 104
10cm
5 x 106
1.25 x 106
5 x 105
1.25 x 105
These are approximate
E63 or CV1 19hr doubling time
Final count on confluent plate
2 days ahead
# cells to seed
3 days ahead
# cells to seed
4 days ahead
# cells to seed
3cm or 6 well
5 x 105
1 x 105
5 x 104
6cm or T-25
1 x 106
2 x 105
1 x 105
10cm
2 x 106
5 x 105
2 x 105
Seeding: 2 day = 48 hr, 3 day = 72 hr
3T3 cells are smaller than E63s so more is needed for seeding
If cell seeding density is known for 10cm dish, for a 6cm dish, divide by 3 and round up.
Useful formula to calculate total cells for final count
2Px = final count on plate
Where 2 represents doubling
x is the initial number of cells
P is the number of periods in time frame
So, in four days for a cell with an 18 hour doubling time, there are 5.3 doubling periods
Tissue culture vessel
Surface growth area
Volume of media
Volume of trypsin
35mm dish
9.0cm2
2.0ml
0.2-0.3ml
60mm dish
21.0cm2
4.0ml
0.5-0.6ml
100mm dish
56.75cm2
10.0-ml
1.0ml
T-25 flask
25cm2
7.0-8.0ml
0.5-0.8ml
T-75 flask
75cm2
20-30ml
1.0ml
T-175 flask
175cm2
45-55ml
2.0ml
6 well multiwell plate
9.6cm2
per well
2.0ml
per well
0.2-0.3ml
per well
12 well multiwell plate
3.8cm2
per well
1.0ml
per well
0.1-0.2ml
per well
24 well multiwell plate
2.0cm2
per well
0.8-1.0ml
per well
0.08-0.10ml
per well
48 well multiwell plate
0.75cm2
per well
0.5-0.8ml
per well
0.05-0.08ml
per well
96 well multiwell plate
0.32cm2
per well
0.1-0.2ml
per well
0.01-0.02ml
per well
I am learning about seeding wells and I am honestly confused with the whole process, such as the calculations, what is a surface area of a 6 well or 24 well plate and how am I supposed to use the information below for this process? Can someone explain what this information means below and how I can use this information when seeding cells in lab? Thank you.
| 3T3 18hr doubling time | Final count on confluent plate | 2 days ahead # cells to seed | 3 days ahead # cells to seed | 4 days ahead # cells to seed |
| 3cm or 6 well | 1 x 106 | 2.5 x 105 | 1 x 105 | 2.5 x 104 |
| 6cm or T-25 | 2 x 106 | 5 x 105 | 2 x 105 | 5 x 104 |
| 10cm | 5 x 106 | 1.25 x 106 | 5 x 105 | 1.25 x 105 |
These are approximate
| E63 or CV1 19hr doubling time | Final count on confluent plate | 2 days ahead # cells to seed | 3 days ahead # cells to seed | 4 days ahead # cells to seed |
| 3cm or 6 well | 5 x 105 | 1 x 105 | 5 x 104 | |
| 6cm or T-25 | 1 x 106 | 2 x 105 | 1 x 105 | |
| 10cm | 2 x 106 | 5 x 105 | 2 x 105 |
Seeding: 2 day = 48 hr, 3 day = 72 hr
3T3 cells are smaller than E63s so more is needed for seeding
If cell seeding density is known for 10cm dish, for a 6cm dish, divide by 3 and round up.
Useful formula to calculate total cells for final count
2Px = final count on plate
Where 2 represents doubling
x is the initial number of cells
P is the number of periods in time frame
So, in four days for a cell with an 18 hour doubling time, there are 5.3 doubling periods
| Tissue culture vessel | Surface growth area | Volume of media | Volume of trypsin |
| 35mm dish | 9.0cm2 | 2.0ml | 0.2-0.3ml |
| 60mm dish | 21.0cm2 | 4.0ml | 0.5-0.6ml |
| 100mm dish | 56.75cm2 | 10.0-ml | 1.0ml |
| T-25 flask | 25cm2 | 7.0-8.0ml | 0.5-0.8ml |
| T-75 flask | 75cm2 | 20-30ml | 1.0ml |
| T-175 flask | 175cm2 | 45-55ml | 2.0ml |
| 6 well multiwell plate | 9.6cm2 per well | 2.0ml per well | 0.2-0.3ml per well |
| 12 well multiwell plate | 3.8cm2 per well | 1.0ml per well | 0.1-0.2ml per well |
| 24 well multiwell plate | 2.0cm2 per well | 0.8-1.0ml per well | 0.08-0.10ml per well |
| 48 well multiwell plate | 0.75cm2 per well | 0.5-0.8ml per well | 0.05-0.08ml per well |
| 96 well multiwell plate | 0.32cm2 per well | 0.1-0.2ml per well | 0.01-0.02ml per well |
