How can Prism do two-way ANOVA with more than 256 replicates per group?

Prism 6 and later allows you to enter up to 256 replicates per data set, in side-by-side subcolumns.

Prism 4 and 5 allow you to enter up to 52 replicates per data set, in side-by-side subcolumns.

Prism 2 and 3 were limited to 16 subcolumns.

If you have more replicates than that, enter the data onto a table formatted for mean, SD (or SEM), and N. If all the N values are equal (or nearly so), ordinary two-way ANOVA will work fine. If the N values vary a lot, the results will be based on an approximation.

You cannot perform repeated measures two-way ANOVA, of course, without entering the raw data, and entry of raw data in Prism is limited by the maximum number of subcolumns. If you have more than 52 (Prism 5) or 256 (Prism 6) subjects you cannot use Prism to perform repeated measures two-way ANOVA.

If you are not performing two-way ANOVA, then consider entering data onto a column table, with replicates stacked rather than side-by-side.