May 27, 2026
Detailed adherent cell passaging protocol
- Ainsley Lederer1
- 1University of Pittsburgh
Protocol Citation: Ainsley Lederer 2026. Detailed adherent cell passaging protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn51mqg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2026
Last Modified: May 27, 2026
Protocol Integer ID: 314203
Keywords: cell growth, cell viability, cell number, doubling time, U2OS, detailed adherent cell, based cell line, u2os cell, cell line, cell, outs of regular cell, regular cell, densities in this protocol, passaging protocol, protocol this protocol, overall protocol, protocol, general protocol, hemacytometer, seeding density
Abstract
This protocol details the ins and outs of regular cell passaging, including how to count cells in a hemacytometer, how to calculate seeding density, and any additional considerations that general protocols may not go over. Concentrations and seeding densities in this protocol are customized to U2OS cells, however the overall protocol can be applied to any adherent-based cell line.
Guidelines
The amount of cells to seed will depend on your cell line, their estimated doubling time, and the total assay time. Read up beforehand on your cell line to get a general idea of seeding densities.
Materials
1. MTT powder (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
2. DPBS buffer
3. Complete growth medium (DMEM, RPMI, others) - ideally you also have phenol red-free media, though it is not strictly required
4. SDS powder
5. HCl (concentrated, or diluted)
6. 96 well TC-treated plate
Cell harvesting
Ideally, you will harvest cells when they are around 70-80% confluence, to prevent stress on the cells. Aspirate the media and wash 2x with PBS.
Add trypsin-EDTA (concentration will vary by cell line) and incubate at 37C for required time (again, time will vary). For U2OS cells, we use 0.1% trypsin with 1 mM EDTA for 1.5 minutes at 37C.
Check under microscope to see whether cells have "rounded up" and begun to release. If not, incubate for 1 minute longer. If they have, "smack" on sides of flask/dish to release cells further, and quickly add complete growth media at 4X the volume of trypsin added (i.e. for 1 mL trypsin add 4 mL media).
Note
For closed flasks this "smacking" protocol is much easier, as you don't need to worry about splashing the media out of the plate. For dishes, tap more gently or be cautious to not spill the media. Rely more on the next step to release cells.
Using a 1 mL pipette, "blow" any cells that are stuck to the plate off. Use a serological pipette to transfer to a 15 mL conical tube. Add a few more mL media to flask/dish, swirl to collect remaining cells, and add to the same 15 mL tube.
Use the serological pipette to vigorously pipette up and down ~10-20 times. This step is necessary to break up cell clumps.
From here, you can either spin the cells down for 5 minutes at 300xG or alternatively count cells directly in step 8. Whether you spin down or not will depend on whether the cells are too dilute or not in the current volume.
If spun down, aspirate the media, leaving behind ~100 uL so as to not disturb the cell pellet. Add fresh media to the tube (volume will depend on cell harvest and flask size). Use the 1 mL pipette to vigorously pipette up and down, again breaking up cells clumps and fully re-suspending pellet.
Cell counting
Take 10 uL of cells into a sterile microcentrifuge tube.
Note
If the cell suspension has been sitting a while, invert the tube several times to ensure even cell distribution.
Add trypan blue - the volume depends on desired dilution factor. If cells are dilute in media, add 10 uL trypan blue (2x dilution). If cells are more concentration, add 40 uL or 90 uL trypan blue (5X and 10X dilution, respectively).
Pipette or flick the tube to ensure the cells are evenly mixed. Load a hemacytometer. For our lab's hemacytometer, 8.5 uL, delivered using a P200 pipette tip, is used.
Note
The P200 pipette tip is used (with a P10 pipette) because larger cells may get stuck in a P10 pipette tip.
Calculate how many cells you have using the following formula:
Use the above formula to calculate how many cells per mL you have. To find the total number of harvested cells, simply multiply this value by your current supension volume. Note that you can also calculate viability by counting the number of blue (dead) cells and dividing by the total number of cells (live and dead). But usually I skip this step unless I see a ton of dead cells.
Now it is time to seed the cells. Use the following chart to decide on how many cells you will seed, then calculate the volume to dispense into the new plate/flask/dish using the cell density (cells/mL) that you calculated in step 9.
| U2OS Cells | Surface area (cm^2) | Seeding density for 20-40% confluence (total cell #) | Cells at 70-80% confluency | vol. trypsin* | vol. media | |
| DISHES | ||||||
| 35 mm | 8.8 | 0.1 x 106 | 0.5 to 1 x 106 | 0.3 mL | 2-3 mL | |
| 60 mm | 21.5 | 0.2 x 106 | 1 to 2 x 106 | 0.5 mL | 4-6 mL | |
| 100 mm | 56.7 | 0.5 x 106 | 2 to 4 x 106 | 1 mL | 8–10 mL | |
| 150 mm | 145 | 0.8 x 106 | 4 to 5 x 106 | 1.5 mL | 15+ mL | |
| FLASKS | ||||||
| T-25 | 25 | 0.25 x 106 | 1 to 2 x 106 | 0.5 mL | 3-5 mL | |
| T-75 | 75 | 0.75 x 106 | 4 to 6 x 106 | 1 mL | 10-15 mL | |
| T-175 | 175 | 1.75 x 106 | 8 to 15 x 106 | 2.5 mL | 25-40 mL | |
| T-225 | 225 | 2.25 x 106 | 15+ x 106 | 3 mL | 40+ mL | |
| PLATES | ||||||
| 6-well | 9.6 | 0.1 x 106 | 1.2 x 106 | 300 uL | 2-3 mL | |
| 12-well | 3.5 | 0.03 x 106 | 0.5 x 106 | 200 uL | 1-2 mL | |
| 24-well | 1.9 | 0.01 x 106 | 0.24 x 106 | 150 uL | 0.5-1 mL | |
| 48-well | 1.1 | 0.03 x 106 | 0.12 x 106 | 100 uL | 200-400 uL | |
| 96-well | 0.32 | 0.01 x 106 | 0.04 x 106 | 30 uL | 100-200 uL | |
| 384-well | 0.084 | 0.0018 x 106 | 0.0072 x 106 | 10 uL | 50 uL | |
| *always neutralize your trypsin with 4x volume of complete media! |
For example, if you harvested cells at a density of 1x10^6 cells/mL and you want to seed a 100 mm dish, take 0.5 mL of your cell suspension (which gives 0.5x10^6 cells) and add to 8 mL media in dish.
Make sure your cells are evenly distributed in the plate by using the north/south, east/west seeding technique.