Fluorescent dye; PKH26 does not bind to a specific molecular target. Its mechanism of action is based on the non-specific, stable integration of its lipophilic aliphatic tails into the lipid bilayer of cell membranes, including plasma membranes and organelle membranes. It partitions into the lipid region without covalent bonding to membrane proteins, enabling uniform labeling of the entire cell surface. This physical insertion mechanism allows PKH26 to label virtually any cell type containing a lipid membrane, including eukaryotic cells, bacteria, parasites, and exosomes.

1. Preparation of PKH 26 Working Solution
1.1 Preparation of Stock Solution
Dissolve 1 mg of PKH 26 in 1 mL of DMSO to obtain a 1 mM stock solution.
Note: It is recommended to store the stock solution at -20°C and -80°C protected from light, and to avoid repeated freeze-thaw cycles.

1.2 Preparation of PKH 26 Working Solution
Dilute the stock solution with serum-free cell culture medium or PBS to obtain a working solution at a concentration of 5-10 µM.
Note: Adjust the concentration of the PKH 26 working solution as needed.

2. Cell Staining
2.1 Suspension Cells (6-well plate)
a. Centrifuge at 1000g for 3-5 minutes at 4°C, discard the supernatant, and wash twice with PBS, 5 minutes each time. The cell density should be 1×10⁶/mL.
b. Add 1 mL of the working solution and incubate at room temperature for 10-45 minutes.
c. Centrifuge at 400g for 3-4 minutes at 4°C, then discard the supernatant.
d. Wash twice with PBS, 5 minutes each time.
e. Resuspend the cells in serum-free cell culture medium or PBS. Observe under a fluorescence microscope or by flow cytometry.

2.2 Adherent Cells
a. Culture adherent cells on sterile coverslips.
b. Remove the coverslip from the culture medium and aspirate off excess medium.
c. Add 100 µL of the working solution, gently shake to ensure complete coverage of the cells, and incubate at room temperature for 5-30 minutes.
d. Wash twice with culture medium, 5 minutes each time. Observe under a fluorescence microscope or by flow cytometry.

Storage
Store at -80°C for 1 year, protected from light.
Precautions
1. Adjust the concentration of the PKH 26 working solution according to actual experimental conditions.
2. This product is for research and development use only and must not be used for drug, household, or other purposes.
3. For your safety and health, please wear a lab coat and disposable gloves during handling.

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PKH-26 is a lipophilic fluorescent dye that stably integrates into the cell membrane without disturbing surface marker expression. It does not impair the viability of labelled target cells. The percentage of apoptotic or PI-positive cells in target cells remained unchanged 4 hours after loading with PKH-26. The expression of surface antigens required for functional effector/target interactions, including CD54, CD58, HLA-ABC, HLA-DR, CD33, CD34, Mel-1, and Mel-2, showed no significant changes 4 hours after PKH-26 staining. Labelling with PKH-26 remains stable in all vital cells tested for a minimum of 72 hours without any loss of fluorescence intensity. Apoptotic target cells exhibit a slightly decreased PKH-26 intensity but can be clearly discriminated as PKH-26-positive target cells.[1]

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In vitro studies demonstrate that PKH26 effectively labels various cell types with high efficiency (typically >99%) without significantly affecting cell viability, proliferation, or biological activity. MTT assays confirmed that PKH26 is cytocompatible with osteoblastic cells and does not exert negative influence on cell growth. For exosome labeling, PKH26 (100 μM) can efficiently label exosomes when incubated at 37°C for 10 minutes. However, coculture experiments have shown that PKH26 can transfer to unlabeled cells via cell debris, indicating that the dye does not remain exclusively within originally labeled cells. In neural stem cell studies, PKH26 (5 μM) labeled 100% of cells at day 1, though viability and proliferation were transiently reduced at day 1 but normalized by day 7.

PKH26 demonstrates remarkable in vivo stability, with fluorescence detectable for up to 100 days or more, making it suitable for long-term cell tracking studies. In murine models, PKH26-labeled human peripheral blood lymphocytes (huPBL) injected intravenously showed rapid distribution, with circulation kinetics monitored over 24 hours. In rat calvarial defect models, PKH26-labeled osteoblasts implanted on scaffolds showed no apparent signs of toxicity in the bone defect area. In gastric cancer xenograft models, PKH26-labeled CIK cells achieved 99.37% labeling efficiency, and tumor growth was significantly inhibited following local, intravenous, or intraperitoneal administration. However, studies have revealed that PKH26 can transfer to host tissues via labeled cell debris, which may lead to false-positive identification of transplanted cells.

PKH26 is not designed for cell-free enzyme/receptor binding assays as it requires lipid membranes for integration. For exosome labeling (a cell-free application), the following protocol is recommended: Exosomes are incubated with 100 μM PKH26 dye solution (50 μL PKH26 dye solution per 100 μg exosomes) at 37°C for 10 minutes in a dark environment. Subsequently, 20 mL PBS is added to stop the staining reaction. The PKH26-labeled exosomes are obtained through differential centrifugation and resuspended in 500 μL PBS for downstream applications. For red blood cell labeling, PKH26 stably incorporates into the erythrocyte membrane lipid portion, enabling the preparation of labeled probes for phagocytosis assays.

For the PKH-26 cytotoxicity assay, target cells (e.g., human melanoma cell lines, primary leukemic AML blasts, or T2 cells) are transferred to tubes and washed twice with serum-free medium. The cells are resuspended in a loading buffer and incubated for 40 minutes at room temperature with freshly prepared PKH-26 (stock 1 mM in ethanol). For loading tumor cells, 0.8 µL PKH-26 is used for small cells (lymphocytes) and 0.5 µL in 200 µL loading buffer, with a final optimal concentration of 2 µM. The dye solution is added slowly while agitating to avoid overstained cells. After 30–40 minutes of shaking at room temperature, the staining is stopped by incubating with human serum for 30 seconds at room temperature. The cell pellet is then transferred to a fresh tube and washed twice with RPMI containing 10% human serum.
After coincubation of PKH-26-labelled target cells (5×10³ cells/well) with effector cells in 96-well V-bottom plates for a recommended standard incubation time of 3 hours, cells are harvested. One mL PBS is added and cells are centrifuged. Cells are then resuspended in 100 µL high calcium annexinV-binding buffer. Staining with 5 µL annexinV-FITC is performed for 10 minutes at room temperature in the dark. Immediately before flow cytometric analysis, 0.1 µg propidium iodide per 100 µL binding buffer is added.
For analysis, target cells are selected by gating on the PKH-26-positive cell population. The percentage of specific cytotoxicity is calculated using the formula: (% specific ann positive cells) = ([ann positive cellssample - ann positive cellscontrol] / [total eventssample - ann positive cellscontrol]) × 100. Concurrent PI staining was found not to add to the information obtained with annexinV-FITC staining alone, as PI single-positive target cells are rare.[1]

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A standard PKH26 cell labeling protocol is as follows: Cells are harvested, washed, and resuspended in Diluent C (provided in the kit) at 2×10⁷ cells/mL. PKH26 dye is diluted in Diluent C to 2×10⁻⁶ M (2 μM). Equal volumes of cell suspension and dye solution are mixed rapidly and incubated for 2-5 minutes at room temperature. The staining is stopped by adding an equal volume of serum or 1% BSA for 1 minute. Cells are then washed three times with complete culture medium to remove unbound dye. For tumor cell labeling, a 40-minute incubation with freshly prepared PKH26 (0.5-0.8 μL of 1 mM stock in 200 μL loading buffer) at room temperature is recommended. Labeled cells can be used for proliferation studies, coculture experiments, or flow cytometry analysis. Labeling efficiency typically exceeds 99%.

A typical in vivo PKH26 cell tracking protocol: Cells are labeled with PKH26 as described above, washed thoroughly, and resuspended in sterile PBS at the desired concentration (e.g., 10×10⁶ cells/200 μL). The labeled cell suspension is administered via the selected route: intravenous (tail vein), intraperitoneal, or local injection (peritumoral or intramyocardial). For tracing studies, animals are sacrificed at predetermined time points (e.g., 2 weeks post-transplantation), and target tissues (e.g., liver, tumor) are harvested for frozen sectioning. Tissue sections are examined under fluorescence microscopy to detect PKH26-positive cells (Ex/Em=551/567 nm). Immunofluorescence staining for specific markers (e.g., albumin for hepatocyte differentiation) can be performed on the same sections. In SCID mouse models, blood samples are collected at various time points (10 min, 30 min, 60 min, 24 h) to assess circulation kinetics.

PKH26 exhibits unique pharmacokinetic properties due to its membrane integration mechanism. The dye demonstrates remarkable in vivo persistence, with detectable fluorescence lasting up to 100 days or longer after labeling. Following intravenous injection of PKH26-labeled cells, circulating cells show rapid initial distribution, with approximately 50% of cells remaining in circulation at 30 minutes and about 30% at 60 minutes post-injection. By 24 hours, a small fraction of labeled cells remains detectable in peripheral blood. The dye integrates stably into cell membranes and is not rapidly cleared or metabolized. However, upon cell death and disintegration, PKH26-containing debris can be phagocytosed by host cells, leading to dye transfer and potential false-positive signals in non-transplanted cells. There is no significant dye leakage from live cells or transfer between intact cells under normal conditions.

PKH26 is generally considered to have low cytotoxicity. In vitro studies confirm that PKH26-labeled cells retain normal biological activities, proliferative capacity, and viability comparable to unlabeled controls. MTT assays and alamar blue assays demonstrated that PKH26 does not inhibit proliferation and shows no toxicity to adipose-derived stem cells compared to unlabeled controls. However, some transient effects have been reported: at 5 μM concentration, PKH26 reduced viability and proliferation at day 1 post-labeling, but these effects normalized by day 7. Over-labeling can lead to loss of membrane integrity and cellular recovery. In vivo, no apparent signs of toxicity were observed in implanted bone defect areas with PKH26-labeled osteoblasts. Delayed cytotoxicity has been reported for other dyes (e.g., Hoechst 33342) but not for PKH26. Overall, PKH26 is considered safe for cell tracking applications when used at recommended concentrations.

[1]. The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity. Methods. 2003;31(2):135-142.

PKH-26 is used as a fluorescent tracer for target cells in a flow cytometric cytotoxicity assay to distinguish between target and effector cells. It is a reliable alternative to the standard ⁵¹Cr release assay, avoiding the use of radioactive isotopes. The PKH-26 assay allows direct evaluation of target cell death (early apoptosis, membrane damage) on a single cell level. The assay showed a good correlation with the ⁵¹Cr release assay (r² = 0.9777), though results from the PKH-26 assay revealed a slightly higher percentage of cytotoxicity. The assay can be completed in as little as 1.5–3 hours. Blocking the perforin pathway with concanamycin A completely blocks the cytotoxic activity measured in the PKH-26 assay. The frequency of antigen-specific T cells (e.g., measured by tetramer staining) does not necessarily correlate with the cytotoxic activity measured by the PKH-26 assay.[1]

C59H97IN2

961.319370031357

960.669

C, 73.71; H, 10.17; I, 13.20; N, 2.91

154214-55-8

162642097

Brown to reddish brown solid powder

0

2

36

62

1200

0

C([N+]1=C(C(C)(C)C2C=CC=CC1=2)C=CC=C1C(C2C=CC=CC=2N1CCCCCCCCCCCCCC)(C)C)CCCCCCCCCCCCCCCCCCCCC.[I-]

XOOOYQALQHJRCY-UHFFFAOYSA-M

InChI=1S/C59H97N2.HI/c1-7-9-11-13-15-17-19-21-22-23-24-25-26-27-28-30-32-34-36-42-51-61-55-47-40-38-45-53(55)59(5,6)57(61)49-43-48-56-58(3,4)52-44-37-39-46-54(52)60(56)50-41-35-33-31-29-20-18-16-14-12-10-8-2;/h37-40,43-49H,7-36,41-42,50-51H2,1-6H3;1H/q+1;/p-1

2-[(E,3E)-3-(3,3-dimethyl-1-tetradecylindol-2-ylidene)prop-1-enyl]-1-docosyl-3,3-dimethylindol-1-ium;iodide

PKH 26; PKH-26; PKH26; 154214-55-8;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 1.67 mg/mL (1.74 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)