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.
Note: It is recommended to store the Cy5-N3 storage solution at -20°C or -80°C in the dark after aliquoting.
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.