Jota arrived for the second-round clash at Molineux but was then left out of the matchday squad altogether as Nuno's side lost 1-0 courtesy of substitute Jacob Brown's late strike.
Jota was thought to have left the ground before kick-off and asked about the issue post-match, Nuno chose not to say whether it was tactical or injury-related.
"He was not available for the game," said the Wolves chief.
"We have a short squad and all the options are good, but him and Max Kilman were not available for this game.
"He wasn’t available, these are decisions we have to make. We have to prepare the squad, have to know what is the moment of the season.
"It was a big turnaround, we have to take care of the players, improve our squad and our team and he was not available for the game. These are our decisions to make."
And on whether Jota stayed for the match or not, Nuno said: "I was watching the game. He was not on the squad list.
"There was no confusion. When a player is not in the squad list sometimes they see the match, if they are away, they stay at home.
"It’s because we have a bubble, we have to make a list, all those details. Nothing strange."
Finally on the subject, asked if Jota will be available for Monday's Premier League clash with Manchester City, Nuno added: "I don’t know. We have to see.
"You are going to speculate a lot. He was not on the squad list. Period."
Ultimately, it was a disappointing night for Nuno as he named a strong XI against the Championship outfit, with five keeping their places from the 2-0 win at Sheffield United.
The £35million record signing Fabio Silva was among those who came in for his gold and black bow, along with compatriot Vitinha who also made his full debut.
Silva had a big chance to score in the second half but put it wide from close range before Brown's shot beat John Ruddy, despite the keeper getting a hand to it, in the 87th minute.
"The first half, I think we should have produced more. We had a lot of the ball but didn't break the lines of Stoke," said Nuno.
"In the second half, it was better and we created more as we pressed better. But it was disappointing.
"We created a couple of chances and we worked hard, but there are a lot of things to improve.
"When you are not well organised, you suffer. And in the moment of the goal, we were not well organised.
"Let's recover from this one and prepare for the next one because it's going to be very tough for us."
Among the few positives on the night were the showings of Silva and Vitinha, and Nuno added: "Of course there are things they can improve, they have to know each other better, get better, but they worked well, Vitor and Fabio, they were available, creating lines of passing, trying to win the game.
"Fabio had a couple of good moments in the box, good movements, next time he will try to be more creative. We will try to work on that, those chances can be better finished.
"Him and Vitor are very young, joined us just this season, it’s about them knowing their team mates better, us trying to create better movement, better solutions because they are talented boys.
"It’s always the same process, to integrate into the team so we can take advantage of the options we have."