This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare working solution: Use a suitable buffer (such as HHBS or PBS) to dilute the TO-PRO 1 stock solution with a concentration of 5 mM and prepare a working solution of 1-5 μM.
Notice:
①When using the stock solution for the first time, it is recommended to pack it into small sizes according to the single dosage and freeze it in the dark at -20℃ or -80℃ to avoid repeated freezing and thawing;
②It is recommended to set the dye solution concentration gradient during the first experiment to determine the optimal working concentration;
③High dye concentration may cause non-specific staining of other cellular structures;
④Please adjust the concentration of the working fluid according to the actual situation and prepare it now.
2. Cell staining (taking 6-well plate as an example)
2.1 Cell suspension staining
(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.
(2) Wash the adherent cells twice with PBS, add trypsin to digest the cells, and centrifuge at 1000g for 3-5 minutes after digestion is completed.
(3) Add 1 mL of TO-PRO 1 dye working solution preheated at 37 °C to resuspend the cells, and incubate at 37 °C in the dark for 15-60 minutes. The optimal culture time for different cells is different.
(4) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, and add PBS for washing 2-3 times.
(5) Resuspend the cells in buffer and observe through fluorescence microscopy or flow cytometry.
2.2 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 100 μL of dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at 37 °C in the dark for 15-60 minutes.
(4) Aspirate and discard the dye working solution, wash the coverslip 2 to 3 times with culture solution, and observe with a fluorescence microscope.
3. Microscope detection: The maximum excitation/emission light of TO-PRO 1 after binding to nucleic acid is 515/531 nm.
Precautions:
1) Fluorescent dyes all have quenching problems. Please avoid light as much aspossible to slow down fluorescence quenching.
2) For your safety and health, please wear a lab coat and disposable gloves.
References:
[1]. Elena Gorokhova, Lisa Mattsson, Annica M Sundström. A comparison of TO-PRO-1 iodide and 5-CFDA-AM staining methods for assessing viability of planktonic algae with epifluorescence microscopy. 2012 Jun;89(3):216-21. doi: 10.1016/j.mimet.2012.03.005. Epub 2012 Mar 14.
