Hi, I have been trying to stain numerous cell types (THP-1, HL60, MV4-11), with a set of lectins (PNA, SNA, MAL-II) all FITC labelled, from Vector Laboratories. I have been incubating at 4 degrees with lectins in PBS for 40 min, at a range of at 1-20ug/mL, and I see almost no fluorescence change from unstained controls, with similar intensity from all lectins. I am concerned that this is just non-specific binding and the lectins aren't binding to their target moieties at all.
I see in some articles there is supplementation with calcium and/or magnesium chloride for staining. Though many don't supplement (or at least don't mention that they do), and still have successful binding. If anyone has a recommended buffer for lectin staining please let me know.
Kindly check https://www.researchgate.net/publication/264799119_Basic_Procedures_for_Lectin_Flow_Cytometry
Dear Logan,
From your description I cant tell what can be the problem, so I can just write down how we did similar experiments.
We used FITC-conjugated lectins (SNA, LCA, PHA-L, MAL-II, ConA, WGA, from Vector Lab) for labelling PBMC. We used 3x105 cells per sample and labelled it in 100 uL FACS buffer (PBS supplemented with 1% FBS and 0.1% sodium-azide) for 20 minutes at 4 Celsius in a range of 1-5 ug/mL of lectin. This was working well for both paraformaldehyde and live cells. Among all the tested lectins, SNA and MAL-II binding was relatively small.
Good luck
Logan Zettle I recommend you to incubate the lectins at room temperature for 40 minutes in TBS or HBS buffer. Supplementing the buffer with calcium or manganese chloride is optional for improving lectin binding. I suggest adding 0.1 mM CaCl2 for PNA, SNA, and MAL II.
To troubleshoot non-specific binding, you may run a negative control by preabsorbing the lectin with its inhibiting sugar and incubating it in place of the unabsorbed lectin.
I hope your problem is solved by now, if not try the buffer listed below. Usually different types of lectins have different metal ion requirements.
Lectin staining buffer - 10 mM HEPES, 150 mM NaCl, 0.1 mM CaCl2
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