You have a confluent T75 of a cell line called A51. These cells are similar in growth and splitting properties to CX cells, except that the double every two days instead of every day. You would like to seed a six-well dish (total growth area - 60 square cm) so that the wells are confluent in one week. What fraction of your cell suspension will you use to seed the six-well dish?
You have a confluent T75 of a cell line called A51. These cells are similar in growth and splitting properties to CX cells, except that the double every two days instead of every day. You would like to seed a six-well dish (total growth area - 60 square cm) so that the wells are confluent in one week. What fraction of your cell suspension will you use to seed the six-well dish?
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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 |