PKH26 Red Fluorescent Cell Linker Kit

Cat. No.: P266287

Overview

The aladdin™ Fluorescent Cell Linker Kits use proprietary membrane labeling technology to stably incorporate a yellow-orange fluorescent dye with long aliphatic tails (aladdin™) into lipid regions of the cell membrane.1,2 The labeling vehicle provided in the kits (Diluent C) is an aqueous solution designed to maintain cell viability, while maximizing dye solubility and staining efficiency during the labeling step. Diluent C is iso-osmotic for mammalian cells and contains no detergents or organic solvents, but also lacks physiologic salts and buffers. The appearance of labeled cells may vary from bright and uniform to punctate or patchy, depending on the cell type being labeled and the extent to which membrane internalization occurs after labeling. However, aladdin™ fluorescence is independent of pH within physiologic ranges and fluorescence intensity per cell is typically
unaffected by the pattern of dye localization.
aladdin™ fluoresces in the yellow-orange region of the spectrum (see Figure 1) and has been found to be useful for in vitro and in vivo cell tracking applications in a wide variety of systems. aladdin™ is compatible with violet, green, red, or far-red viability probes used in cytotoxicity assays and with fluorescent proteins, antibodies, or DNA dyes emitting in these spectral regions. One of the most common uses of aladdin™ is proliferation analysis based on dye dilution. Applications of dye dilution proliferation analysis include estimation of antigen-specific precursor frequencies and identification of quiescent or slowly dividing stem or progenitor cells in normal and neoplastic tissues.16-20 aladdin™ has also proven useful for monitoring: uptake of exosomes, viruses, platelets, and other nanoparticles; apportionment of membrane in stem cell division; cell-cell membrane transfer；phagocytosis; antigen presentation; adhesion; signaling through gap junctions, and neuronal migration in tissue slices. Due to its extremely stable fluorescence, aladdin™ is the cell linker dye of choice for in vivo cell tracking studies, particularly when labeled cells are to be followed for periods longer than a few weeks.
Components:

Components	P266287-0.1ml	P266287-1ml
B:aladdin™ dye	0.1ml	10ml
B:Dilution solution C	10ml	60ml

Precautions:
● The working solution of the dye is ready for use. Do not store the prepared dye to affect the dyeing effect.
●During the aladdin™ staining process, there must be no azides or metabolic toxicants.
●Although adherent cells can also be stained, a single cell suspension is best for uniform staining. Therefore, the staining effect is better after digesting adherent cells into single suspension cells with protease (trypsin/EDTA).
●Remove serum and lipid before staining to improve staining effect.
●The presence of salt can cause the dye to form particles and interfere with the dyeing reaction. Therefore, it is important to resuspend the cells before adding the dye. The dye should be added directly to the cell suspension, not to the cell mass.
● Excessive cell labeling will result in loss of membrane integrity and decreased cell number. The cell and dye concentration in this sample is a reference concentration suitable for most cells, but the best dye/cell is determined by the user according to the cell type and experimental purpose. In addition, the user must also evaluate the cell viability (iodine excretion), fluorescence intensity, and fluorescence. Coefficient of variation of peak value, uniformity of dyeing.
● The stain concentration varies according to the type of cell and the number of cells in each well. The number of generations or times that can be traced after different cell types are marked is quite different. Please make a test based on the actual situation or reference documents.
Operation Note: Fast and uniform mixing is very important for marking. The following measures should be taken to obtain the best results:
1) The cell suspension and working dye solution should be the same amount when mixing;
2) Avoid staining too much (>5ml) or too little (<100ul) liquid. Avoid adding dye with a pipette stained with serum;
3) The liquid volume should be as precise as possible to ensure that the cell and dye concentration are accurately replicated.
4) The time that the dye and diluent act on the cells is as short as possible and has certain toxicity. The diluent can be used to act on the cells according to the above steps to see the degree of damage.
5) Add the same amount of serum to terminate the reaction. Do not centrifuge the cells in Diluent C before terminating the staining reaction. Use serum-containing medium to increase the cleaning effect.
6) The cells should be transferred to a new tube and centrifuged separately, and the culture medium should be used instead of Diluent C when washing 3 times.
7) Ideal for marking in vitro stem cells, lymphocytes, monocytes, endothelial cells, etc.
8) Whole cell labeling should precede the labeling of monoclonal antibody staining. The cell tracking probe will remain stable when the monoclonal antibody is stained at 4°C; if the labeling is followed by monoclonal antibody staining, the phenomenon of "blocking" is likely to occur.
9) The stained cells are fixed with 2% paraformaldehyde and the stability is more than 3w.
Materials needed:
Uniform single cell suspension; serum-containing medium; serum-free medium or PBS without Ca2+Mg2+; serum, albumin or protein source compatible with the medium; polypropylene conical centrifuge tube; temperature controllable Centrifuge (0-1000g); fluorescence analyzer (fluorometer, fluorescence microscope, flow cytometer, fluorescence image analyzer); ultra-clean table; cell counter; glass slide.

Operation steps:
1. General cell membrane markers
Lipophilic dyes bind to the cell membrane to complete labeling. The dye intensity is a function of dye concentration and cell concentration, and is not related to permeability. Therefore, it is crucial to ensure that the amount of dye added is not excessive. Over labeled cells will result in loss of membrane integrity and/or reduced cell viability.
The following processes can be used for labeling cells both in vivo and in vitro, including lymphocytes, monocytes, monocytes, neurons, or any other small cells
Cell. The labeling process of cells in the body needs some improvement, such as staining of small plates or selective labeling of phagocytic cells.
The cell concentration and dye concentration during the following dyeing process represent the initial concentration of the operation. This concentration has been proven to be applicable to a variety of cells. Users need to evaluate the cell viability (such as PI staining), fluorescence intensity, staining uniformity, and whether it affects the function of the studied cells after staining, and determine the optimal dye concentration and cell concentration based on the experimental objectives. Aseptic operation demonstration steps (total volume 2ml, final staining concentration 2 × 10-6M aladdin) ™  Dye and 1 × 107 cells/ml, all operations at 20-25 ℃)
1. Cells are digested with trypsin and/or EDTA to form a single-cell suspension. 2 × 107 cells are placed in a conical centrifuge tube and washed once with sterile culture medium.
Attention: White egg whites and lipids will bind to dyes, reducing the concentration of dyes bound to the cell membrane. It is best to wash the cells once with a blank medium or buffer before resuspending them with diluent C (step 4).
2. Form loose cell clusters at 400 × g for 5 minutes away from the heart.
be careful: aladdin ™  Dyes cannot be directly added to the sediment, as this can cause uneven staining and reduced cell viability.
After leaving the heart, the heart aspirates and discards the upper clear fluid, leaving less than 25ul of fluid on the cell mass.
Note: To obtain reproducible experimental results, it is important to reduce the volume of residual culture medium or buffer when resuspending with diluent C.
4. Add 1mL of diluent C and gently mix with a pipette to prepare a 2 × cell suspension. Resuspend the cells to ensure complete dispersion, do not shake, and do not store the cells in diluent C for too long.
Attention: The presence of physiological salts can cause dye clumping and significantly reduce dyeing efficiency. Ensure that the cells are suspended in diluent C at the time of staining, without any culture medium or buffer salt.
Before dyeing, add 4 μ L of aladdin ™  Add 1mL of alcohol solution to diluent C, mix thoroughly, and prepare 2 x dye solution (4 x 10-6M).
Note 1: To reduce the effect of alcohol on cell viability, the dye added in step 5 should ensure that the final concentration of alcohol in step 6 does not exceed 1-2%.
Note 2: If the final concentration of the required dye is less than 2 × 10-6M, 100% alcohol should be used to add aladdin ™  Dilute in a separate container to ensure reproducibility of experimental results.

6. Quickly add 1mL of 2 × cell suspension (step 4) to 1mL of 2 × staining solution (step 5), and mix the sample evenly and quickly using a pipette immediately, as uniform staining occurs instantaneously. The final cell concentration is 1 × 107/mL, aladdin ™  The concentration is 2 × 10-6M.
Attention: Due to the instantaneous completion of staining, it is crucial to quickly mix the cells with the dye to obtain bright, uniform, and reproducible labeling results. To achieve the best results, the following measures should be taken:
a. Do not use aladdin ™  Add the dye directly to diluent C containing 2 x cells.
b. Mix 2 x cell suspension (step 4) with an equal volume of 2 x dye solution (step 5).
c. Adjust the concentration of 2 x cells and 2 x dye to avoid staining volumes that are too small (<100 μ L) or too large (>5mL).
d. Quickly mix cells and dye with an electric pipette. The mixing speed of the cleaning pipette is too slow, resulting in uneven staining. The mixing of oscillation and vortex oscillation is also slow, and the uniformity of dyeing is poor.
e. Allocate volumes as accurately as possible to ensure reproducibility between samples and experiments.
7. After mixing, the stained cells were incubated at 25 ℃ for 2-5 minutes, and the centrifuge tube was gently inverted at regular intervals to ensure thorough mixing at 25 ℃. Due to the fast dyeing speed, extending the incubation time is not helpful for the experiment.
Attention: Keep the cells in the dye solution for as short a time as possible while ensuring the desired dye intensity. Due to the lack of physiological salts in diluent C, prolonged exposure to diluent C can cause a decrease in certain cell viability. If the degree of its impact cannot be determined, a control group that is only diluted and a control group that only uses alcohol without dye can be added.
8. Add an equal volume of serum (2mL) or an equal amount of serum or 1% BSA for staining reaction, and incubate for 1 minute to bind excess dye.
Note 1: Qingqing (or equivalent egg white concentration) is the optimal final solution. If using complete culture medium (including clear tissue culture medium) as a substitute, add a volume of 10mL.
Note 2: Do not terminate the reaction by adding diluent C or centrifugation.
Note 3: Do not use blank culture media or buffer salts, as they can cause dye aggregation. The aggregation of dyes makes it impossible to clean during the cleaning process, resulting in the presence of unlabeled cells during the analysis process.
9. Leave the cells at 400 × g for 10 minutes at 20-25 ℃, and carefully aspirate the supernatant. Resuspend 10mL of complete culture medium (including clear tissue culture medium), transfer the resuspension to a new sterile centrifuge tube, and incubate at 400 × g for 5 minutes at 20-25 ℃. Wash twice with 10mL of complete culture medium to remove unbound dye.
Note 1: Transfer the heavy suspension to a new centrifuge tube to reduce the impact of residual dye on washing efficiency; Note 2: Do not clean with diluent C.

10. After the final step of cleaning, resuspend the cells in 10mL of complete culture medium to evaluate cell recovery rate, cell viability, and fluorescence intensity. The required vitality of suspended cells in the heart
Cell concentration.
Note 1: Stained cells can be fixed with neutral formaldehyde, and under dark conditions, the fluorescence intensity should remain stable for at least 3 weeks.
Note 2: The fluorescence intensity of the dye is generally 100-1000 times that of the background. Although the CV value of the dye is related to the type of cell, the fluorescence distribution should be as uniform and symmetrical as possible.
11. Fluorescence microscopy/flow cytometry analysis of cells. Check the cell recovery status, passage status, and fluorescence concentration. The dye should be uniform, with a fluorescence level 100-1000 higher than the background
Twice.
aladdin ™  Dyeing optimization

MC-38 TIL cells were treated with the specified aladdin mentioned above ™ Concentration staining resulted in a final cell concentration of 1 × 107/ml. The vitality (▲) was measured by FITC staining, and the average fluorescence intensity (●) was detected by flow cytometry. ⽤20μM aladdin ™ After labeling, there was no change in the specificity and efficacy of anti-tumor TIL.
2. Histology:
When preparing and storing PKH labeled cell sections, frozen sections and special sealing specimen techniques are required.
Cutting preparation:
1. After removing the tissue, it was immediately frozen with ice.
2. Store at -70 ℃ before cutting.
3. Prepare frozen sections using OCT (Tissue Tek; Miles, INC.) complex.
4 4-5 μ m tissue cutting.
5. Dry the glass at room temperature for 1 hour.
6. Use 1-2 drops of cyanoacrylate adhesive ester glue to seal and cover the glass.
7. Use standard filters such as FITC (PKH2 and PKH67) or TRITC (aladdin) for inspection and photography ™）。
Double dyeing cutting:
1. Soak the glass in acetone solution for 24-48 hours and remove the cover;
2. Wash with distilled water to remove acetone;
3. Easy to use Mayer or Harris spirit for dyeing and cutting;
4. Sealing and fixing glass with AS/AP permanent water-based solid-liquid sealing (Bio/Can America, Inc., Porland, ME)
Attention: Due to the fact that organic solvents can extract PKH staining solution, while counterstaining absorbs fluorescence, simultaneous observation of histology and PKH fluorescence is not advisable.

Steps

Special Reminder:
1) All products of our company are only for professional use in life science research and cannot be used for clinical diagnosis or treatment, food or medicine, or stored in ordinary residential areas.
2) All products of our company must be operated by qualified professional technicians while wearing masks/gloves/lab coats and complying with the safety operating procedures of the biological laboratory!

Storage and Shipping

Storage

Protected from light,Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads