CY5-N3

Guide (The following is our recommended protocol. It is provided as a guide only and should be modified to suit your specific needs).
1.1 Preparation of storage solution^[3]
Use anhydrous DMSO to prepare Cy5-N3 stock solution.
1.2 Preparation of working solution
Use preheated serum-free cell culture medium or PBS to dilute the storage solution to prepare a 100 μM Cy5-N3 working solution.
Note: Please adjust the concentration of Cy5-N3 working solution according to the actual situation and prepare it before use.
2 Specific staining steps
1) Incubate Yersinia pseudotuberculosis YPIII (pIB102; pFU95) cells in calcium-deficient LB medium at 26°C for 1 h and then at 37°C for 2 h to induce the T3SS with or without the addition of test compounds.
2) Dilute the bacterial suspension 1:5 with PBS and transfer 50 μL to microfluidic chambers (CellASIC ONIX B04A-03 plates).
3) Prepare the click reaction mixture: add 0.1 mM CuSO₄ (HY-Y1878), 128 μM TBTA (HY-116677), 5 mM Sodium ascorbate (HY-B0166A), and 100 μM Cy5-N3 to PBS.
4) Wash the chambers with PBS at a flow rate of 4 psi for 10 min.
5) Inject the click reaction mixture into the chambers at a pressure of 8 psi for 5 min and then at 4 psi for 60 min to complete the conjugation reaction.
6) Remove excess reagents by washing with PBS at 8 psi for 10 min and 4 psi for 20 min.
7) Analyze the stained bacteria using a confocal spinning disk microscope (far red 647 laser) to detect the fluorescent signal.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.