u/nebulalegacy

Hi everyone, I'm currently running a 2nd biological replicate using an 11-color spectral flow cytometry panel on leukemia cell lines to track surface receptor density during forced differentiation. Everything is going fine, but I have hit a hard wall with my CD-135 (FLT3) inventory.
I have exactly enough CD-135 left for my remaining 80 samples if I use 2.5 µL per sample with absolutely zero waste. Because I normally use a 10% volume overage for my master mixes (to account for pipette dead volume), including CD-135 in the daily cocktail means I will mathematically run out before the end of the project. I can't order a replacement in time.

My proposed workaround:
For today’s run (16 tubes total):

1. I calculate and mix the 10% overage cocktail for the other 7 antibodies + Immunofluorescence Buffer, completely omitting the CD-135.

2. I pipette 47.5 µL of this CD-135-free master mix into each cell pellet

3. I take a P10 pipette and manually spike exactly 2.5 µL of pure CD-135 directly into each of the 16 individual tubes, changing tips every time and striking off the tubes to ensure I don't miss any.

My questions for the experts:

1. Will manually spiking this one antibody alter the binding kinetics or MFI reproducibility compared to a fully pre-mixed cocktail, assuming the final volume (50 µL) and incubation time (45 mins at room temp in the dark) remain identical?

2. Has anyone else resorted to this manual spiking method to survive a low-reagent situation, and did it skew your data?

I know it increases the risk of technical pipetting error, but it seems like the only mathematically sound way to get to the finish line. Any advice or validation would be hugely appreciated!

I'm a first-year PhD student running multiparametric flow cytometry (10-color, CytExpert) on 5 cell lines with 1 untreated control and 7 drug conditions and 3 biological replicates.

I forgot to include an antibody-unstained control tube for any of my 5 cell lines in Replication 1.

A labmate has unstained data run on the same cytometer using the same instrument template and identical voltages. However, the unstained was run with all 5 cell lines mixed together in one tube, not as separate single cell line tubes.

My questions:

1. Is a mixed unstained control usable as a baseline reference for single cell line experiments, or does the blending of different autofluorescence profiles make it unreliable?

2. My plan going forward is to run a proper single cell line unstained for each cell line in Replicates 2 and 3, and use those as the reference for Replicate 1 as well. Since unstained controls reflect instrument settings and autofluorescence rather than biological variables. Is this reasoning sound?

3. Any other suggestions for how to handle the missing unstained in Replicate 1?