u/RiccardoCecchin

I'm trying to enrich for cells that have stably integrated a transposon construct using FACS. Unfortunately, our construct has no fluorescent or other expression marker, so we're relying on two cell-surface markers, CD81 and CD47, that should be overexpressed following transposition. The problem is that both markers are already constitutively expressed in our target cells, and we don't know the transposition efficiency or how much their expression will increase following integration.

I've performed antibody titrations for both markers, but I'm unsure how best to determine the optimal staining conditions for sorting.

My understanding is that the usual goal of an antibody titration is to identify the lowest antibody concentration that provides good separation between negative and positive populations, with minimal background and sufficient staining of the positive population.

However, in my case, I'm not trying to distinguish truly negative cells from positive cells. Instead, I'm trying to distinguish cells with normal endogenous expression from cells with increased expression due to stable transposition.

Wouldn't it make more sense, in this case, to use an antibody concentration that preserves the differences in fluorescence intensity between cells with normal and elevated expression, rather than one that saturates the epitopes? If the antibody concentration is too high and staining approaches saturation, could that compress the fluorescence differences between normal and high-expressing cells and make them harder to distinguish?

Or am I misunderstanding this?

I'm also unsure how best to optimise the sorting strategy when I don't know the expected increase in CD81 or CD47 expression, or the proportion of cells that have successfully integrated the construct.

Since we're using a transposon system rather than lentiviral transduction, increasing the MOI isn't an option for improving separation.

I'd appreciate any advice, particularly from anyone who has experience sorting cells based on relatively small increases in endogenous cell-surface marker expression rather than using a distinct positive/negative marker.

Hi Reddit, I’d like some advice regarding whether I should send a final text to someone who seems to be ghosting me.

A couple of weeks ago I went on a solo trip overseas and met an amazing local woman during a night out. We immediately clicked and started dancing, talking, kissing and ended up spending the night together. What followed was basically a 4-day romance where we saw each other every chance we had.
We went on dates, long walks, kayaking trips, scenic spots around the city, shared breakfasts, stayed in hotels together, took photos everywhere, and spent a lot of time just talking and being affectionate. She even asked me to be her boyfriend at one point. I told her I was genuinely interested in trying to make things work despite the distance, especially since I may soon move to a nearby country for work.
What confuses me is that her behaviour during those days felt incredibly genuine. She would skip trains just to keep talking to me longer, hold onto me in public, and make time for me despite being busy with exams. It didn’t feel casual or one-sided at all.
After I left, though, her communication slowly faded with first slower and shorter replies and now essentially silence just a few days after I left.
She still occasionally interacted through Setlog (for those familiar with the app), but stopped replying properly to messages and has now left me on read.
I know short travel romances can feel more meaningful to one person than the other, but I genuinely struggle to believe she felt nothing given how much time and emotional energy she invested while I was there.
At this point I’m considering sending one final calm message, not to pressure her, but just to close the loop respectfully and acknowledge that the experience meant something to me.
Do you think that’s a bad idea? And if not, how would you structure that kind of message without sounding needy or emotional?
Thank you for reading.