Principle and precautions of CFDA and SE cell proliferation tracking fluorescent probe detection
CFDA-SE can be used for cell tracking and also for cell proliferation detection. Cells labeled with CFDA-SE can be used for in vitro and in vivo proliferation studies, and have the function of not staining adjacent cells. CFDA-SE is most commonly used for lymphocyte proliferation detection, and can also be used for proliferation detection of other cells such as fibroblasts, natural killer cells, hematopoietic progenitor cells, etc.
Detection principle
CFDA SE can be broken down into CFSE by intracellular esterases after entering cells through intact cell membranes. CFSE can randomly and irreversibly bind to lysine residues or other amino groups in intracellular proteins, and label these proteins. CFDA-SE labels cells with green fluorescence. The fluorescence of the progeny cells labeled with CFDA-SE decreases by half every time they divide. The fluorescence intensity of the cells that divide once is halved (1/2), and the fluorescence intensity of the cells that are separated twice is halved again (1/4), and so on. The fluorescence intensity gradually decreases in the order of 1/2, 1/4, 1/8, and 1/16.
usage method
1. Preparation of preservation solution (5 mM)
Prepare as needed, such as taking 360 μ L of DMSO, dissolving 1 mg of CFSE, and sonicating to obtain 5 mM CFSE preservation solution. Divide it into 40 μ L volumes and place it in a light shielded sterile cryovial. It can be stored at -20 ℃ for 2 months.
2. Preparation of working fluid
Take 1 μ L of 5mM CFSE diluent and dilute it with 1mL of 10% FCS PBS.
3. Dyeing
(1) Resurrected cells. Resuspend cells in PBS with 5% FCS at a concentration of generally 1x1x10 ^ 6/mL (in vitro experiments) or 1x1x1x10 ^ 7/mL (transfection experiments).
(2) Staining. Mix 1mL DFSE working solution with 1mL cell suspension and incubate at 37 ℃ for 5-10 minutes. Gently mix twice during the process.
(3) Terminate staining. Wash three times with complete culture medium and centrifuge. Generally, the third wash is performed after incubating for 5 minutes after the second wash. Cold containing 10% newborn bovine serum, RPMI-1640 medium (containing L-glutamine) (item number: AC01L064）， Gently mix for 1 minute (bounce gently with your hand, avoid blowing), centrifuge at 1500 r/min for 5 minutes.
(4) Flow cytometry is used for detection in FL1 channel.
matters needing attention
✔ All reagents must be thoroughly thawed and mixed before use, and efforts should be made to avoid the formation of bubbles during the mixing process; CFDA SE solvent will solidify at lower temperatures. Please take a water bath at 20-25 ℃ for a moment until completely melted before use.
✔ The reagent contains fluorescent dyes. When storing or labeling, try to avoid light exposure to avoid fluorescence quenching issues.
✔ The lactonase activity varies among different cells, resulting in differences in staining effects. The optimal working concentration can be explored based on cell type and culture conditions.
✔ If cell fixation is required, use aldehyde fixatives such as 3.7% paraformaldehyde to fix at room temperature for 15 minutes; If additional labeling such as antibody labeling is required later, please permeabilize the cells with ice acetone for 10 minutes.
✔ For your safety and health, please wear lab coats and disposable gloves when operating.
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