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LumiCell PKH26 cell membrane labeling kit manual

Kit components

Kit component Count
13201
100 uL dye, 1x buffer
23201
100 uL dye, 5x buffer
33201
500 uL dye, 1x buffer
43201
500 uL dye, 5x buffer
2484-100uL, PKH26 dye, 1 mM solution in isopropanol, 100 uL 1 1 5 5
K6150, PKH26 Diluent, 1x, 10 mL 5 25
K7150, PKH26 Diluent, 5x, 10 mL 1 5

Store at +4°С. Warm to RT before use.

Shelf life 12 months.

Recommendations for using the kit

  • Optimal concentrations of the dye and cells can vary depending on cell and study type, so evaluate cell viability, homogeneity and fluorescence intensity after staining.
  • Do not use azide-containing solutions when staining with PKH26 dye.
  • Staining is more homogeneous when cell suspension is used.

Protocol

Protocol for cell membrane labeling with PKH26 for RAW264.7 adhesion culture, 1×106 cells/sample, the final concentration of PKH26 2 µM, final volume 200 μL.

  1. Prepare PKH26 solution immediately before staining. Add 1 µL of 1mM PKH26 solution to 9 µL of 96% ethanol, add 4 µL of the resulting solution to 100 µL of PKH26 Diluent, 1x.
  2. Remove the cell culture from the surface with a scraper in Hanks’ solution (HBSS). Count the cells in the sample. Add 3 mL of Hanks’ solution, сentrifuge at 400g for 6 min at room temperature.
    *Serum proteins and lipids also bind the dye, so it is recommended to wash the cells once with serum-free medium or phosphate buffer saline.
  3. Remove the supernatant with a pipette, resuspend a necessary amount of cells (e. g. 106 cells) in 100 µL of PKH26 Diluent, 1x. Add 100 µL of PKH26 solution prepared during step 1. Pipette and allow to stand at room temperature for 5 min. The final PKH26 concentration in the cell solution is 2 µM.
    *To obtain reproducible results, minimize the volume of the supernatant before cell resuspending.
    *Do not leave cells in PKH26 Diluent for a long period.
    *Staining is almost instant, so rapid cell dispersion in the dye solution is important to produce bright homogeneous and reproducible labeling.
  4. Add 2 mL of fetal bovine serum to stop the reaction, incubate for 1 min. Centrifuge at 400g for 40 min at room temperature.
    *To stop the reaction, do not use a serum-free medium or buffered salt solution that results in dye aggregates.
  5. Remove the supernatant, resuspend the cells in 5 mL of complete culture medium, transfer to a new tube. Take aliquots to evaluate cell viability with trypan blue. Centrifuge at 400g for 40 min at room temperature.
  6. Resuspend the cells in a buffer for further analysis (microscopy, flow cytometry etc.).
    *Stained cells can be fixed with 2% paraformaldehyde, and staining remains stable for at least 3 weeks if samples are protected from light.

Recommendations for storage

PKH26 solution can be stored at room temperature or in a refrigerator protected from light. Check the solution for precipitation before use. If a precipitate is seen in the dye solution, slightly warm it up on a water bath at 37°C and ultrasonicate or vortex until redissolved.

PKH26 Diluent is delivered as a 1x or a 5x solution in a sterile container. Store in a refrigerator and adjust to room temperature immediately before use.

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