Rhodamine B (Basic Violet 10)

This plan only provides a guide, please modify it to meet your specific needs.

1. Solution preparation

(1) Stock solution: Dissolve Rhodamine B in DMSO to prepare a stock solution with a concentration of 10mM.
Note: It is recommended that unused stock solution be stored at -20°C or -80°C in the dark after aliquoting to avoid repeated freezing and thawing.

(2) Working solution: Dilute the stock solution with experimental buffer to the required working concentration.
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.

2. Cell suspension staining (taking 6-well plate as an example)

(1) Suspended cells are centrifuged at 1000g for 3-5min. Discard the supernatant and wash twice with PBS, each time for 5min.

(2) Adherent cells are washed twice with PBS, trypsin is added to digest the cells, and after digestion, they are centrifuged at 1000g for 3-5min.

(3) Add 1mL Rhodamine B working solution to resuspend the cells and incubate at room temperature in the dark for 5-30min. The optimal culture time varies for different cells.

(4) After incubation, centrifuge at 1000g for 5min, remove the supernatant, and wash with PBS 2-3 times, 5min each time.

(5) Resuspend the cells in serum-free cell culture medium or PBS and observe by fluorescence microscopy or flow cytometry.

3. Cell adhesion staining

(1) Culture adherent cells on sterile coverslips.

(2) Remove the coverslip from the culture medium, aspirate the excess culture medium, and place the coverslip in a humid environment.

(3) Add 100µL Rhodamine B working solution from one corner of the coverslip, gently shake to evenly cover all cells with the dye, and incubate at room temperature in the dark for 30-60min.

(4) Aspirate the dye working solution and wash the coverslip 2-3 times with culture medium, 5min each time. Observe by fluorescence microscopy.

4. Labeling of Antarctic Candida lipase B (CALB) with Rhodamine B Steps^[1] (from the literature, for reference only)

(1) Prepare 5mg/mL Rhodamine B solution in DMSO and take 0.4mL.

(2) Prepare 5mg/mL CALB solution in Na₂CO₃-NaHCO₃ buffer (50mM, pH 9) and take 4mL.

(3) Add 0.4mL of Rhodamine B solution to 4mL of CALB solution and stir the mixture at 4°C for 12h.

(4) Terminate the reaction with 50mM NH₄Cl.

(5) Dialyze the reaction mixture against PBS (50mM, pH 7) at 4°C for 48h to remove unreacted dye.

References:
[1] Xu W, Fu Z, Chen G, et al. Graphene oxide enabled long-term enzymatic transesterification in an anhydrous gas flux[J]. Nature communications, 2019, 10(1): 2684.