I use Lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 647 Conjugate (Cat No. L32460), 1mg/ml, to stain germinal center B cells in murine splenocytes. With dilutions of 1:100, 1:500 and 1:1000, I do see that all splenocytes are positively stained, but the intensity increases as I increase the concentration of the reagent used. I never see a bi-modal or a broad distribution of PNA even in stimulated cells (with 1:100). Any thoughts on the 100 percent staining being real or if there are any precautions to be used to prevent non-specific binding of PNA?
Hello Devi.
Use of lectins for cell labelling could be a nightmare if you are not using a fresh, newly-obtained stock with an inhibitory sugar for the carbohydrate specificity as a control group in which abolish the cell labelling.
Specially old lectins stored inappropriately have intact fluorecein-conjugated lectin molecules, degraded free lectin molecules, and free fuorescein molecules that can stain whole cells throughout the cytoplasm due to the bacterial growth.
If your stock is not new, get a new one, Add 4 mg BSA/ml PBS (milipored) to lectin sol to make 500ug/ml lectin, preventing loss of lectin, serve as a carrier protein, and prevent non-specific binding. Aliquot ~5-10ul in tubes, store at -70C until use. Do all these procedure under lamina flow.
Upon use, dilute with staining buffer, centrifuge, and use the upper fraction.
Do not forget the staining tube containing specific oligosaccharide and lectin together, and then cell staining as a control group. This will save your time and money.
The other concern is the way you collected cells from the spleen. If you used harsh treatment of protease, it may most likely expose cell surface proteins by excessive digestion, leading to sticky exposed peptides (even the non-glycoproteins). Thus you need to negotiate mild digestion with cell numbers. Last words: just check the stained cells under fluorescence microscope whether the lectin labels only the cell surfaces, it should. The final concentration may lie at 1~5ug/ml
Best wishes.
Dr Lee,
Thank you for the answer. The PNA I use is less than 6 months old. I have resuspended the lyophilised powder in MilliQ water along with sodium azide and stored aliquots at
Hi Devi,
I suspect your lectin has gone off. Frequent openings of freezer will degrade FITC-lectin through frequent freezings and thawings. It would take quite a while to drop temperature again once you opened, causing thawing until it freezes again.
To verify whether your PNA lectin staining is specific to the specific glycoconjugates on the cell surfaces. Thus, co-incubation of excessive sugar concentration and lectin for 30 min. This co-incubated lectin will be used to stain the cells, negative staining will be expected by the excessive competitive bindings of the sugars to the lectin instead of to the cell surface glycoconjugates.
You'd better to get a new PNA, I can definitely say. They should be stored at -70, once thawed, the remaining solution should be thrown away without sitting down at 4C. The staining procedure and operation of taking lectin solution are probably done under non-sterile condition in every lab. NaN3 is not a solid solution once a large number of bacteria are inoculated during operation. Take my advice!!! Get a new one.
Cheers.
Hello Devi,
Could you solve your problem? If so please give me some advices. I'm also facing similar kind of problem. I use FITC Peanut Agglutinin (Vector, cat no. FL-1071), 5mg/ml to stain germinal center B cells in murine splenocytes. I diluted PNA at 2.5-20 microg/ml concentrations in 0.5% BSA in 10mM HEPES, 150mM NaCl, 2mM EDTA, 0.09% NaN3 (staining buffer) to stain 1 million cells in 100 microl. I also found more than 80% cells are stained positive with PNA at each concentration (2.5-20 microg/ml). Notably, I use fresh stock of PNA. I'm not sure if my staining procedure is correct or not as I couldn't find any specific detail protocol of PNA flow cytometry.
If you share your knowledge and experience with me on this matter, it could help to overcome my trouble. Thank you.
Hi Salman,
I haven't been able to lay hands on a new vial of PNA. Nor have I resolved the issue. I thought I'll use GL7 instead. But no progress as of now.
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