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TROUBLE SHOOTING GUIDE FOR PKH2, PKH26 AND PKH67 FLUORESCENT CELL LINKER DYES

Problem Solution

Cell Clumping Dye concentration too high. Lower labeling concentrations.

Serum not added to stop reaction. Serum, other protein or complete medium must be used to stop labeling reaction.

Platelets present in whole blood sample. Centrifuge sample at low speed to remove platelets before staining.

Adherent cells not disaggregated. Mechanical (aspirate through needle) or enzymatic (trypsin) digestion prior to staining.

Poor initial cell viability of sample. Incubate with 0.002% DNase for 30 minutes at 37^oC. before staining.

Cells exposed to dye too long. Expose cells to dye for 2-5 minutes.

Poor Staining Intensity Filters incorrect for observing dye. Check filter set-up. Check cell pellet after labeling; if pellet is pink or yellow, cells are stained.

Working dye stock prepared too long before adding to cells - dye aggregating. Prepare dye immediately prior to labeling.

Salt content of labeling solution too high - dye aggregating. Centrifuge cells and remove as much supernatant as possible to minimize physiological salts after suspension in Diluent C.

Serum present during labeling interfering with dye incorporation. Wash cells in serum-free buffer 1-2 times prior to resuspension in Diluent C for labeling.

Staining intensity varies with cell type based on cell size/surface area, lipid to protein ratios of membranes and lipid component of membranes. Adjust dye concentration for each cell type and experimental application
Analyze sub-populations independently.

Dye concentration too low. Increase dye concentration.

Cell concentration too high. Reduce cell concentration to a range of 10⁶ to 10⁹ cells/ml.

Patchy or Punctate Staining Salt content of labeling solution too high - dye aggregating. Centrifuge cells and remove as much supernatant as possible to minimize physiological salts after suspension in Diluent C.

Some localization of dye on certain cell membranes causes a patchy appearance. May be cell type dependent and not fixable.

Hetero geneous Staining Different cell types in sample - staining intensity varies from cell type to cell type. Isolate desired cell types prior to labeling - perform analysis on one cell type only.

Heterogeneous exposure to dye during staining. Ensure rapid and homogeneous mixing at the time of dye addition .
Optimize order of addition and method of mixing.

Dye concentration too low. Increase dye concentration.

Cell concentration too high. Lower cell number per ml, use a range of 10⁶ to 10⁹ cells/ml.

Serum present during labeling interfering with dye incorporation. Wash cells in serum-free buffer 1-2 times prior to resuspension in Diluent C for labeling.

Dye sticking to test tube walls. Use only polypropylene tubes, other plastics absorb dye.

Cells clumped - not single cells in suspension. Carefully suspend cells in media - use aspiration through a needle to break clumps.

Cells Dead Dye concentration too high. Lower dye concentration.

Serum used to stop reaction was not heat inactivated. Heat serum at 56^oC for 60 minutes.

Sensitivity to diluent. Run diluent only control - monitor viability.

Viability/ Function Poor viability or recovery.
Functional alteration of cells.
Over incorporation of dye molecules (over-labeling) will affect cells - adjust (lower) dye concentration and/or (increase) cell concentration.

Difficulty resuspending pelleted cells because of clumping in bottom of test tube. Over incorporation of dye molecules (over-labeling) will affect cells - adjust (lower) dye concentration and/or increase cell concentration.
Incubate cells with 0.002% DNase for 30 minutes at 37^oC after stopping staining and before centrifuging.

Cells appear shriveled when visualized in microscope. Non-mammalian cells may require special diluent formulations - Call Sigma Technical Service.

Dye Leakage/ Transfer Dye transfer between cells in co-culture. Wash cells 3-5 times after labeling - transfer sample to new tubes between washes. PKH2 tends to transfer more readily between cells in co-culture - try PKH67 or PKH26.

Leakage of dye from target cells in cytotoxicity assays. PKH2 tends to transfer more rapidly from certain cell types (e.g. K562, NS-1 cell lines) - try PKH67 or PKH26.

Organic based solvents or detergents will extract dye. Use only aqueous-based fixatives and solvents.

Phagocytosis of target cells by effector cells. Monitor for phagocytosis by microscopy.

Transfer occurs initially, but stops over time. Culture in medium for several hours prior to mixing with unlabeled cells [Embleton, M.,et al., Int. J. Cancer, 49, 566 (1991)].

Fixation of tissue sections Frozen sections show no fluorescence. Use only dry ice to freeze tissue for sectioning - other solutions (i.e. isopentane, DMSO) will extract dye.

Low fluorescence intensity. Be sure sections are dry before adding glue - allow glue to dry.
Check autofluorescence of tissue and then select appropriate cell linker dye.

Counterstaining absorbs fluorescence. Use serial sections or use a single section and perform microscopy before demounting and counterstaining for morphology.

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TROUBLE SHOOTING GUIDE FOR PKH2, PKH26 AND PKH67 FLUORESCENT CELL LINKER DYES
	Problem	Solution
Cell Clumping	Dye concentration too high.	Lower labeling concentrations.
	Serum not added to stop reaction.	Serum, other protein or complete medium must be used to stop labeling reaction.
	Platelets present in whole blood sample.	Centrifuge sample at low speed to remove platelets before staining.
	Adherent cells not disaggregated.	Mechanical (aspirate through needle) or enzymatic (trypsin) digestion prior to staining.
	Poor initial cell viability of sample.	Incubate with 0.002% DNase for 30 minutes at 37^oC. before staining.
	Cells exposed to dye too long.	Expose cells to dye for 2-5 minutes.
Poor Staining Intensity	Filters incorrect for observing dye.	Check filter set-up. Check cell pellet after labeling; if pellet is pink or yellow, cells are stained.
	Working dye stock prepared too long before adding to cells - dye aggregating.	Prepare dye immediately prior to labeling.
	Salt content of labeling solution too high - dye aggregating.	Centrifuge cells and remove as much supernatant as possible to minimize physiological salts after suspension in Diluent C.
	Serum present during labeling interfering with dye incorporation.	Wash cells in serum-free buffer 1-2 times prior to resuspension in Diluent C for labeling.
	Staining intensity varies with cell type based on cell size/surface area, lipid to protein ratios of membranes and lipid component of membranes.	Adjust dye concentration for each cell type and experimental application Analyze sub-populations independently.
	Dye concentration too low.	Increase dye concentration.
	Cell concentration too high.	Reduce cell concentration to a range of 10⁶ to 10⁹ cells/ml.
Patchy or Punctate Staining	Salt content of labeling solution too high - dye aggregating.	Centrifuge cells and remove as much supernatant as possible to minimize physiological salts after suspension in Diluent C.
Patchy or Punctate Staining	Some localization of dye on certain cell membranes causes a patchy appearance.	May be cell type dependent and not fixable.
Hetero geneous Staining	Different cell types in sample - staining intensity varies from cell type to cell type.	Isolate desired cell types prior to labeling - perform analysis on one cell type only.
	Heterogeneous exposure to dye during staining.	Ensure rapid and homogeneous mixing at the time of dye addition . Optimize order of addition and method of mixing.
	Dye concentration too low.	Increase dye concentration.
	Cell concentration too high.	Lower cell number per ml, use a range of 10⁶ to 10⁹ cells/ml.
	Serum present during labeling interfering with dye incorporation.	Wash cells in serum-free buffer 1-2 times prior to resuspension in Diluent C for labeling.
	Dye sticking to test tube walls.	Use only polypropylene tubes, other plastics absorb dye.
	Cells clumped - not single cells in suspension.	Carefully suspend cells in media - use aspiration through a needle to break clumps.
Cells Dead	Dye concentration too high.	Lower dye concentration.
	Serum used to stop reaction was not heat inactivated.	Heat serum at 56^oC for 60 minutes.
	Sensitivity to diluent.	Run diluent only control - monitor viability.
Viability/ Function	Poor viability or recovery. Functional alteration of cells.	Over incorporation of dye molecules (over-labeling) will affect cells - adjust (lower) dye concentration and/or (increase) cell concentration.
	Difficulty resuspending pelleted cells because of clumping in bottom of test tube.	Over incorporation of dye molecules (over-labeling) will affect cells - adjust (lower) dye concentration and/or increase cell concentration. Incubate cells with 0.002% DNase for 30 minutes at 37^oC after stopping staining and before centrifuging.
	Cells appear shriveled when visualized in microscope.	Non-mammalian cells may require special diluent formulations - Call Sigma Technical Service.
Dye Leakage/ Transfer	Dye transfer between cells in co-culture.	Wash cells 3-5 times after labeling - transfer sample to new tubes between washes. PKH2 tends to transfer more readily between cells in co-culture - try PKH67 or PKH26.
	Leakage of dye from target cells in cytotoxicity assays.	PKH2 tends to transfer more rapidly from certain cell types (e.g. K562, NS-1 cell lines) - try PKH67 or PKH26.
	Organic based solvents or detergents will extract dye.	Use only aqueous-based fixatives and solvents.
	Phagocytosis of target cells by effector cells.	Monitor for phagocytosis by microscopy.
	Transfer occurs initially, but stops over time.	Culture in medium for several hours prior to mixing with unlabeled cells [Embleton, M.,et al., Int. J. Cancer, 49, 566 (1991)].
Fixation of tissue sections	Frozen sections show no fluorescence.	Use only dry ice to freeze tissue for sectioning - other solutions (i.e. isopentane, DMSO) will extract dye.
	Low fluorescence intensity.	Be sure sections are dry before adding glue - allow glue to dry. Check autofluorescence of tissue and then select appropriate cell linker dye.
	Counterstaining absorbs fluorescence.	Use serial sections or use a single section and perform microscopy before demounting and counterstaining for morphology.