Cell Passaging into Multi-well Plates (6, 12, 24, 96-well)
Multiwell plates allow for high-throughput
or parallel experiments. Cells must be evenly seeded at the appropriate
density to ensure consistent results across wells. This protocol covers
proper seeding and scaling down of trypsinization and medium volumes for
different plate formats.
Reagents Required
-
Complete growth medium (e.g.,
DMEM + 10% FBS)
-
1× PBS (without Ca²⁺/Mg²⁺)
-
Trypsin-EDTA (0.05% or 0.25%)
-
70% Ethanol
Equipment
-
Cell culture-treated multiwell
plates
-
Biosafety cabinet (Class II)
-
CO₂ incubator (37°C, 5% CO₂)
-
Inverted microscope
-
Pipettes and sterile tips
Procedure
1. Cell
Preparation (From T25 or Any Source)
-
Follow standard trypsinization
as above.
-
Count cells using a
hemocytometer or cell counter to seed precise cell numbers.
2. Recommended
Seeding Densities & Volumes
|
Plate Type |
Volume per Well |
Typical Seeding Density |
|
6-well |
2.0–2.5 mL |
1,00,000-200,000 |
|
12-well |
1.0–1.5 mL |
50,000-100,000 |
|
24-well |
0.5–1.0 mL |
20,000-50,000 |
|
96-well |
100–200 µL |
2,000-10,000 |
Adjust based on experiment type
(e.g., transfection, drug assay).
3. Plate
Seeding
-
Label the multiwell plate with
cell line name, date, and experiment info.
-
Dilute cells in complete medium
to the required density.
-
Add the calculated volume of
cell suspension to each well using a pipette.
-
Gently rock the plate
front-to-back and side-to-side to distribute cells evenly.
-
Place the plate in a 37°C, 5%
CO₂ incubator.
Notes
-
Avoid over-confluence: passage
before reaching 90–100%.
-
For even seeding in 96-well
plates, use multichannel pipettes.