Jar of Flies

Jar of Flies is an activated item added in The Binding of Isaac: Afterbirth.

Effects[]

Killing an enemy adds a fly to the jar, up to a maximum of 20.
Upon use, releases Blue Flies equal to the number of flies in the jar.
This item belongs to the Beelzebub set. Collecting three items from this set will transform Isaac into a giant humanoid fly.

Kills by blue flies and locusts (including flies from the jar) and shooting familiars (including Incubus) do not add flies to the jar.
Blue spiders, orbital familiars, and chasing familiars do add flies to the jar.
Friendly enemies such as from the Friendly Ball add flies to the jar, but not those spawned from other enemies.
Batteries do not affect the number of flies in the jar.
Enemies must be actually killed to add a fly; transformations (such as Gapers turning into Pacers) or knockouts (such as incapacitating a Globin) do not count as kills.
Destroying a shopkeeper will count as a kill.
With items that kill normally unkillable enemies, killing things like Stone Grimaces will add flies to the jar.
Killing minions in the Hush fight does not add flies to the jar.

Hive Mind: Flies are bigger and deal double damage.
Schoolbag: Flies will be gained even while the Jar of Flies is in the inactive slot.

Book of Virtues: Using Jar of Flies summons wisps instead of Blue Flies; the wisps do not summon the flies when destroyed. Up to five flies at a time are consumed to summon one wisp, with stacking health and damage. The damage of the wisps decreases as they take damage. Enemies killed by wisps’ tears will not add flies to the jar.

Car Battery: No effect.
Uranus: Enemies frozen by Uranus do not grant extra flies, even if shattered. Only enemies killed with bombs will add flies.
Void: Flies will not collect when absorbed by Void, making the interaction worthless.

The item's pickup quote references the tagline of the Pokémon games. The quote said "Gotta catch 'em all?"
With Booster Pack #2, this item's sprite fills up with more flies depending on how many flies it contains. This is based on this Steam Workshop mod by Stewartisme.
The item is likely a reference to the Alice In Chains album of the same name.