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试剂盒组分
试剂盒组分 | 数量 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
13201 100 uL dye, 1x buffer |
23201 100 uL dye, 5x buffer |
33201 500 uL dye, 1x buffer |
43201 500 uL dye, 5x buffer |
14201 100 uL dye, 1x buffer |
24201 100 uL dye, 5x buffer |
34201 500 uL dye, 1x buffer |
44201 500 uL dye, 5x buffer |
17201 100 uL dye, 1x buffer |
27201 100 uL dye, 5x buffer |
37201 500 uL dye, 1x buffer |
47201 500 uL dye, 5x buffer |
||
2484-100uL, PKH26 dye, 1 mM solution in isopropanol, 100 uL | 1 | 1 | 5 | 5 | — | — | — | — | — | — | — | — | |
K6150, PKH Dyes Diluent, 1x, 10 mL | 5 | — | 25 | — | 5 | — | 25 | — | 5 | — | 25 | — | |
K7150, PKH Dyes Diluent, 5x, 10 mL | — | 1 | — | 5 | — | 1 | — | 5 | — | 1 | — | 5 | |
2485-100uL, PKH2 dye, 1 mM solution in isopropanol, 100 uL | — | — | — | — | 1 | 1 | 5 | 5 | — | — | — | — | |
2801-100uL, PKH800 dye, 1 mM solution in isopropanol, 100 uL | — | — | — | — | — | — | — | — | 1 | 1 | 5 | 5 |
保质期 12 个月。
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 PKH dyes.
- Staining is more homogeneous when cell suspension is used.
Protocol
Protocol for cell membrane labeling with PKH dyes for RAW264.7 adhesion culture, 1×106 cells/sample, the final concentration of PKH dye 2 µM, final volume 200 μL.
- Prepare PKH dye solution immediately before staining. Add 1 µL of PKH dye solution (PKH dye, 1 mM solution in isopropanol) to 9 µL of 96% ethanol, and add 4 µL of the resulting solution to 100 µL of PKH Dyes Diluent, 1x. *1x PKH Dyes Diluent is available either in ready-to-use form (K6150, PKH Dyes Diluent, 1x) or as a 5x concentrate (K7150, PKH Dyes Diluent, 5x). To dilute 5x PKH Dyes Diluent, use sterile bidistilled water.
- 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 400 x g 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.
- Remove the supernatant with a pipette, and resuspend a necessary amount of cells (e. g. 1×106 cells) in 100 µL of PKH Dyes Diluent, 1x. Add 100 µL of PKH dye solution prepared in step 1. Pipette and allow to stand at room temperature for 5 min. The final PKH dye 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 PKH Dyes 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.
- Add 2 mL of fetal bovine serum to stop the reaction, and incubate for 1 min. Centrifuge at 400 x g for 10 min at room temperature. *To stop the reaction, do not use a serum-free medium or buffered salt solution that results in dye aggregates.
- Remove the supernatant, resuspend the cells in 5 mL of complete culture medium, and transfer them to a new tube. Take aliquots to evaluate cell viability with trypan blue. Centrifuge at 400 x g for 10 min at room temperature.
- 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
PKH dye 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 in a water bath at 37 °C and ultrasonicate or vortex until redissolved.
PKH Dyes 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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