Pick the format by the experiment, not by habit
Format decides how many cells you can grow, how much media each well drinks, and how forgiving the culture is when something drifts. The right question is what the endpoint needs, not what the lab usually orders.
Every format from 6 well to 96 well grows the same cells. What changes is scale and margin for error. Big wells give you material to harvest and room for the culture to breathe. Small wells give you replicates and throughput, at the price of faster evaporation, smaller samples, and less tolerance for a missed feed. Work backwards from the readout: how many cells does one data point need, and how many days must they stay healthy to get there.
Cell numbers and media volume
As a rough guide, a confluent well holds on the order of 1 million cells in a 6 well plate, a few hundred thousand in 12 well, around 200 thousand in 24 well, and tens of thousands in 96 well, though the real figure swings widely with cell size and line. Working media volumes follow the same ladder: roughly 2 to 3 mL per 6 well, 1 to 2 mL for 12 well, 0.5 to 1 mL for 24 well, a few hundred microliters for 48 well, and 100 to 200 microliters for 96 well. Use these as planning ranges, then confirm against your protocol.
When 96 well culture makes sense
Ninety six well plates shine when the assay is the endpoint: viability and proliferation readouts, cytotoxicity screens, reporter assays, any measurement taken in the well by a plate reader. You get replicates and full dose curves on one plate, reagent use drops, and the culture only needs to stay uniform for the assay window. Seeding can be automated, reads are fast, and the small volumes that make long culture risky are an advantage here, because the experiment ends before evaporation and nutrient depletion start to bend the results.
When 96 well culture fails
Stretch a 96 well culture over many days and the format fights you. A 100 microliter well evaporates measurably each day, fastest at the plate edges, so outer wells drift in concentration and growth while inner wells hold steady. There is little media reserve, so fast growing cells exhaust it and acidify between feeds, and frequent feeding of 96 small wells invites contamination and pipetting error. For long cultures, differentiation, or experiments needing cells harvested at the end, step up to 24 or 12 well and run the final assay in a sample taken from them.
Inserts: culture with two sides
A multiwell insert is a small cup with a porous membrane bottom that hangs inside a well, dividing it into an upper and a lower compartment. Cells grow on the membrane while media and soluble factors pass through the pores, so you can culture two cell types together without mixing them, feed from one side and sample from the other, or model a barrier such as an epithelium and measure what crosses it. Inserts are the standard tool for co-culture, transport, and permeability studies, and they fit the larger formats, most often 12 and 24 well.
Related guides
Common questions
How many cells should I seed per well?
It depends on the line, the well area, and how many days the culture runs before readout. A common starting point for an overnight attachment followed by a short assay is a fraction of confluent density, scaled by growth area (roughly 0.3 cm2 for 96 well up to about 9.5 cm2 for 6 well). Optimize with a small seeding density titration in your own format rather than copying a number from a different plate.
Why do the edge wells of my 96 well plate look different?
Edge wells evaporate faster than inner wells, so media concentrates and cells there grow differently over time. For sensitive assays, fill the outer wells with sterile media or buffer and use only the inner 60 wells for samples, or use plates and lids designed to limit evaporation for long incubations.