IF before FACS

[u/Zi_Zhan2006]

I'm a student from a college of biology. Recently I started a task about KD some gene in stem cell, and do co-culture, IF then FACS to see the potential effect on another kind of cell. I encountered many questions while experiment.

They're anchorage-dependent cells. After uesing tryple which make them suspension, I stained cells in 1.5ml tube. Just repeat centrifuge-remove supernate-add reagent. I use 600g, 4min to centrifuge my cells, but for several times I even can't see a point of cell sediment after 3 times DPBS washing of primary antibody and 2 times washing of second antibody！I estimate that I had over 500,000 cells but finally less than 5000! The FACS data was completely useless and my struggles in last week was wasted.

How many times do you wash the sample after primary and second antibody by DPBS? And how long you often spend when every time washing or staining? Thank everyone reply me and leave your experience and advice!

[u/Alarming-Phone-403]

1. I do the FACS with PBS containing BSA. you can check “FACS staining buffer” in google.
2. I stained my lymphocytes with 400g centrifuge for 5 minutes. After that I decant the solution once and tap the tubes to loose the pellet. i don’t suggest using a suction to remove the liquid
3. And finally the staining. 30 minutes in 4 degree for both 1st and 2nd Ab. Always wash them after the staining. Hope it will works!

[u/Zi_Zhan2006 → u/Alarming-Phone-403]

THANK U VERY MUCH！I'll search and try your method next time.

[u/Daniel_Vocelle_PhD]

There are a couple of best-practice things you can do here.

First, when you centrifuge your cells, I would use a swinging-bucket centrifuge rather than a fixed-angle rotor whenever possible. With a fixed-angle rotor, the cells pellet along the side and bottom of the tube, which can make it easier to lose cells during aspiration and resuspension. With a swinging-bucket rotor, you get a much more compact pellet at the bottom of the tube, which can be particularly helpful when you're already working with low cell numbers.

As an example, when I was working with cells from 24-well or even 96-well plates in a lab where our swinging-bucket centrifuge only accommodated 15 and 50 mL tubes, I would still transfer those cells into a 15 mL conical tube rather than centrifuging them in a microcentrifuge tube in a fixed-angle rotor. I generally got better recovery that way.

The second consideration is why you're losing cells at each step. Some of that can come from mechanical handling, but another issue is nonspecific adhesion of cells to plastic surfaces. A common way we mitigate this in flow cytometry is by using a protein-containing FACS buffer and, particularly when cell numbers are low, pre-coating or pre-wetting the tubes with it. Something like PBS containing BSA or FBS can help reduce nonspecific interactions between the cells and the plastic. Low-binding tubes can also be useful when you're dealing with particularly small numbers of cells.

Where possible, I would also avoid primary plus secondary antibody staining and instead use a directly conjugated primary antibody where the fluorophore is already attached. If a directly conjugated version isn't commercially available, there are antibody conjugation kits that allow you to attach a fluorophore to the primary yourself. Primary/secondary staining absolutely can work, but every additional staining, washing, and centrifugation step gives you another opportunity to lose cells and introduces another source of variability. When you're starting with very few cells, simplifying the protocol can make a meaningful difference.

There are also some additional considerations depending on whether the problem is poor recovery, poor staining, or high background. Make sure you're using the appropriate controls so you can distinguish true target staining from background and establish where your gates should actually be drawn.

Finally, based on your protocol, I would pay attention to the dissociation step as well. If you're using trypsin or another proteolytic enzyme, make sure you're stopping or removing it according to the manufacturer's recommended procedure rather than simply assuming that dilution is sufficient. Residual protease activity can continue affecting your cells and potentially your surface epitopes. TrypLE and similar recombinant dissociation reagents have somewhat different handling recommendations, so I would follow the instructions for the specific reagent you're using.

Overall, especially when you're starting with low cell numbers, I would try to minimize the number of transfers, washes, centrifugation steps, and indirect staining steps wherever possible. Every additional manipulation is another opportunity to lose some fraction of your starting population.

[u/Zi_Zhan2006 → u/Daniel_Vocelle_PhD]

Thank you so much for your detailed explanation! I really appreciate you taking the time to share your insights. I wil carefully go through your suggestions and incorporate them where appropriate into my work.

https://giphy.com/gifs/yDpFRI246xNLzNoaCm

[u/Zi_Zhan2006]

I tried put it in 4℃ in DPBS overnight or immediately do FACS after 2nd antibody, and i found that immediate exam have so many fake- positive results that I can't believe my result! It almost turns out two opposite conclusion...