Sulfo-NHS-LC-Biotin sodium

Protocol for biotinylation of exosome proteins ^[1]:

Sulfo-NHS-LC-Biotin was utilized to tag proteins that are exposed on the external surface of intact exosomes. It features a larger spacer arm measuring 30.5 Å between the biotin molecule and the amine reactive linker. This extended spacer arm is beneficial as it helps overcome steric hindrance, especially in the crowded environment of the exosome surface, leading to improved labeling efficiency.

1. 200μg of intact exosomes were combined with 10 mM Sulfo-NHS-LC-Biotin and incubated at room temperature for 30 minutes.
2. Following the 30-minute incubation, the excess Sulfo-NHS-LC-Biotin was removed by passing the mixture through a 10 KDa MWCO (molecular weight cut-off) filtration device.
3. Biotinylated exosomes were washed with 10× volumes of 1× PBS (phosphate-buffered saline) and concentrated to a final volume of 30 μl. Subsequently, the biotinylated exosomes were lysed using 300 μl of RIPA buffer for 1 hour, followed by three freeze and thaw cycles.
4. After lysis, a buffer exchange was performed to replace the RIPA buffer with 1× PBS. This step was necessary because RIPA buffer contains primary amines that can interfere with the subsequent biotinylation process.
5. The lysed-biotinylated exosomes were then exposed to 10 mM Sulfo-NHS-Biotin at room temperature for 30 minutes to label any remaining free amines. Any excess biotin was subsequently removed.

Protocol for monitor cell Blood-Testis Barrier (BTB) integrity ^[2]:

Note: Freshly prepare a stock of 10 mg/ml Sulfo-NHS-LC-Biotin in PBS/CM pH 8.0 (10 mM sodium phosphate, 0.15 M NaCl) containing 1 mM CaCl₂ as labeling buffer (biotin reagent is freshly prepared immediately before use, within 1–2 h before its use; do not prepare stock solution for storage).

1. Sertoli cells were inoculated into 12-well culture plates and cultured with 3mL DMEM/F-12 medium for 4-5 days to establish functional permeability barrier. The medium is changed every 48 hours and supplemented with growth factors.
2. Wash the cells with cold PBS (pH 7.4) and wash the cells three times to remove the amine-containing medium and protein from the cells.
3. Add 20 μL biotin reagent per mL DMEM/F-12 for biotinization reaction mixture(see note).
4. Add 1 mL of biotinylation mixture to each hole of the 12-well plate. Incubate the reaction mixture at room temperature for 30 minutes.
5. Wash cells three times with PBS + 100 mM glycine to quench the biotinylation reaction, and gently remove excess biotin reagent and byproducts by aspiration.
6. Fix cells in ice-cold methanol at room temperature for 5 min (or cells can be fixed in 4% PFA (paraformaldehyde) in PBS for 5 min, wash thrice in PBS at room temperature, and then permeabilized in 0.1% Triton X-100 for 10 min, this step is not necessary using ice-cold methanol to fix Sertoli cells).
7. Wash with PBS thrice at room temperature. Incubate with blocking solution (1% BSA in PBS, wt/vol) for 30 min at room temperature.
8. Remove blocking solution by aspiration. Add ~50 μL of Streptavidin-FITC (Cat.No.GC19886) containing 4′,6-diamidino-2-phenylindole (DAPI, to visualize Sertoli cell nuclei). Incubate for 1 h at room temperature.
9. Wash with PBS thrice at room temperature. Dry coverslips in dark. Mount onto microscope slides.

Phosphate-buffered saline (PBS)/CM: 10 mM NaH₂PO₄, 0.15 M NaCl, 1 mM CaCl₂, pH 8.0 at 22℃.

References:

[1]. Diaz G, Wolfe LM, et,al. Changes in the Membrane-Associated Proteins of Exosomes Released from Human Macrophages after Mycobacterium tuberculosis Infection. Sci Rep. 2016 Nov 29;6:37975. doi: 10.1038/srep37975. PMID: 27897233; PMCID: PMC5126699.

[2]. Wu S, Wang L, et,al. An In Vitro Assay to Monitor Sertoli Cell Blood-Testis Barrier (BTB) Integrity. Methods Mol Biol. 2021;2367:207-213. doi: 10.1007/7651_2021_390. PMID: 33830455.