There are many small black spots in fetal bovine serum under the microscope. Will there be interference during flow cytometry?

I suggest running a parallel control: take part of the serum, centrifuge first (400-500g for 10 min), and collect the supernatant; then take another portion and filter it directly through 0.22 μm. Prepare culture media separately with each, culture the same batch of mouse splenocytes, and after staining compare the CD4/CD8 ratio and debris rate. If the ratio shows no obvious change after the two treatments, then the effect of the black dots on the experimental results is controllable; if the debris rate is markedly higher and the cell populations show tailing, it indicates that the particles are interfering with gating. Australian serum is not cheap, so do not throw it away all at once. Another key point is the thawing method: overnight at 4°C will cause calcium phosphate and fibrinogen to settle at the bottom of the bottle. Before aliquoting, it should be rapidly rewarmed in a 37°C water bath and gently mixed; when aliquoting, do not pour from the bottom of the bottle. If this batch of serum still has a large amount of particles after filtration, contact the supplier to request the factory mycoplasma and endotoxin data, or ask for a replacement lot.

Before doing flow cytometry, first distinguish the source of the black dots. Mycoplasma is not visible under a standard inverted microscope, let alone appearing as small black dots; the absence of obvious Brownian motion also does not resemble bacterial contamination. It is more likely calcium carbonate/calcium phosphate precipitate from Australian fetal bovine serum during repeated freeze-thaw cycles or aliquoting, with uneven size and many background particles. You can take 2 mL of serum, add it to serum-free 1640 or PBS, culture at 37°C for 48 h, and observe whether it becomes turbid; then send it for mycoplasma PCR confirmation. Black dots alone cannot be used to diagnose mycoplasma contamination. When gating on FSC/SSC, these precipitate particles are usually smaller than lymphocytes, and their signal falls in the very low FSC/SSC region, so they may be mistaken for debris; a large number of particles will raise the background, affect the threshold, and increase the proportion of the debris gate, but they generally will not fall directly into the CD4/CD8 double-positive cell gate. It is recommended to first gate with CD45 or add a live-cell dye, then gate CD3+, and finally examine CD4/CD8.

If it is newly replaced Australian serum and a large number of black dots appear, it is recommended not to rush to discard the entire batch. For splenic lymphocytes, black dots may indeed interfere with gating, especially if the cell suspension is mixed with culture medium supernatant. Before running the samples, wash twice with PBS containing 0.5% BSA and centrifuge at 200-300g for 5 min, which can remove many small particles; when resuspending, do not make the cell density too high to avoid clumping. On FSC/SSC, black dots often appear in the debris region with low FSC and low SSC, sometimes overlapping with apoptotic cell debris. Slightly increasing the FSC threshold or starting gating from CD45+ can exclude non-cellular particles. You can first take an unstained single-positive control tube to see where the black dots actually fall, and then decide whether to adjust the voltage. Filtering serum through 0.22 μm has little effect on lymphocyte culture, but a large amount of precipitate can easily clog the membrane, so it is recommended to filter through 0.45 μm first and then 0.22 μm.