Flow Cytometry for Separation of Keratinocyte Subpopulations from the Viable Epidermis

Haftek, Marek; Viac, J.; Cordier, G; Thivolet, J

doi:10.1111/1523-1747.ep12455541

Cited by 15 publications

(13 citation statements)

References 21 publications

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“…This presumption was further reinforced by the detection of desmosealin within the desmoglea. The terminal differentiation‐dependent expression of this latter protein has been well documented in our previous studies (12,16).…”

Section: Discussionsupporting

confidence: 68%

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

Sandjeu

Callejon

Vincent

et al. 2011

Experimental Dermatology

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Here, we report a method, adapted to the human epidermis, allowing isolation of desmosomes in small tissue fractions. The methods previously developed for animal skin did not work efficiently with human tissue. Enrichment of desmosomes was performed by the association of two incubation steps in acidic solutions containing detergent NP-40 at two different concentrations followed by a sonication step. The suspension was centrifuged twice: first to remove the heavy cell fragments and then at 16 000 g on a discontinuous sucrose gradient. A desmosome-enriched fraction (DsF) was collected at the 30-50% sucrose interface. We demonstrate by immunoelectron microscopy and by western blotting that the central part of the desmosome structure is preserved as well as the antigenicity of its components. Our approach, allowing a significant enrichment of the cell fractions containing desmosomes, can be used to immunize animals and create new antibodies directed against desmosomal components. Using this strategy, new and so far poorly studied molecules incorporated into the desmosome cores could be targeted more easily.

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Section: Discussionsupporting

confidence: 68%

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

Sandjeu

Callejon

Vincent

et al. 2011

Experimental Dermatology

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“…A number of techniques for separating epidermal cells into individual subpopulations have been described [7,15], avoiding serial subcultivations which may alter functional capacities of keratinocytes [9,22]. Here we presented a method for the purification of oral keratinocytes using an immunomagnetic separation technique after two-step enzymatic digestion.…”

Section: Discussionmentioning

confidence: 99%

Magnetic cell separation for purification of human oral keratinocytes: an effective method for functional studies without prior cell subcultivation

Formanek

Temmel

Knerer

et al. 1998

European Archives of Oto-Rhino-Laryngology

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In studying human oral keratinocytes, it would be very helpful to obtain a pure population of cells without prior in vitro expansion. An immunomagnetic separation technique, or magnetic cell separation (MACS), was modified for efficient purification of human oral keratinocytes. Subsequent to two-step enzymatic digestion, the cell suspension was labelled with a mouse anti-CD45 (pan-leukocyte) monoclonal antibody (MoAb) to stain mononuclear cells. In a second step a rat anti-mouse antibody conjugated with colloidal superparamagnetic particles was used. Labelled cells were retained in the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. The unlabelled, unretained cells were further examined by flow cytometry analysis, enzyme-linked immunosorbent assay and polymerase chain reaction. After the MACS procedure, unretained cells showed a strong positivity for the lu-5 MoAb (as a marker for pan-cytokeratin) and were negative for anti-vimentin (to mark mesenchymal cells), for anti-CD45 MoAb and for melanocyte-detecting antibodies, thus representing pure keratinocytes (> 98%). Purified keratinocytes maintained full viability (> 91%) and functional capacities. [3H]thymidine uptake and epidermal growth factor (EGF) receptor expression were unaltered when compared with the non-separated cell population. Furthermore, interleukin-1 alpha was detected at the protein and RNA levels in keratinocytes immediately after MACS enrichment. Our findings show that MACS appears to be a useful tool for purification of oral keratinocytes and allows for further functional studies without prior subcultivation of cells.

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“…at bigh resolution; can be used in combination with image analysis devices To obtain quantitative, compositional information in the context of its position within the tissue [133][134][135]; primary method used with x-ray microprobe and energy dispersive x-ray microanatysis; also used to obtain data on the mass of a structure using linear density measurements of tbe imaged structure, e.g., mass of intermediate filaments [97] (Continued on p. 87S) To reveal information about the molecular structure of crystalline objects thereby to assist in understanding molecular interactions; has been used to demonstrate periodic patterns of extracted, stretched and unstretched keratin [20,[92][93][94]; reconstituted keratin filaments [95], keratin from patients with harlequin ichtbyosis [136], stretched crystalline lipid in stratum corneum cells [137], and to show that the lipid patterns characteristic for the stratum corneum reflect tbe total composition ofthe lipids in the intercellular space [138] The epidermis and dermis can be separated at the dermal-epidermal junction by a variety of methods (beat, enzymes, etc. ); controlled separation ofthe epidermis below the granular layer can be accomplished by the injection of staphylococcal epidermolytic toxin [64]; the epidermal sbeets and dermis tben can be used independently in experiments and studied by various morphologic techniques Celts can be separated enzymaticaily and sorted with a cell sorter [139], or by panning, rosetting (e.g., for Langerhans cells), density gradient or velocity sedimentation, or by sequential trypsinization. Isolated preparations are easily pelleted and embedded and sectioned or affixed to slides for microscopic examination.…”

Section: [] []mentioning

confidence: 99%

Biologic Structure and Function: Perspectives on Morphologic Approaches to the Study of the Granular Layer Keratinocyte.

Holbrook¹

1989

J Invest Dermatol

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S tephen Rothman [1] wrote: "Modern dermatology has been built on the solid pillars of precise macro-and microscopic observation, accurate recording and meaningful interpretation ofthe findings. No natural science can exist without direct observation of natural phenomena, and dermatology has become a great discipline because we have had so many good observers." He went on to state further that "... whereas the immense knowledge acquired by the classical descriptive methods is still rapidly increasing, the application of experimental methods to dermatologic problems is a relatively young and undeveloped approach." These statements acknowledge the importance of observation and description (usually thought of as features of morphology) in clinical dermatology and in descriptive dermatologic research, but were made before he could recognize the value of morphology to experimental studies as well. Unfortunately, Dr. Rothman did not see the remarkable expansion in dermatologic research that has taken place within even the last 20 years, an eta in which descriptive biology and experimental biology are not necessarily separate entities or even parallel concepts. Morphology is simply one of many approaches --sets of tools -that can be used to solve problems in biology.Morphology is often the starting point of an investigation. Understanding the structure of a cell or tissue provides a framework on which other kinds of data can be applied. A morphologic study, however, need not be synonomous with description and qualitative investigation. Many ofthe techniques used by morphologists provide compositional information (presence/absence of a chemical compound) on a structural substrate (location within the cell or tissue) which, in turn, permits interpretation of function. For example, electron microscopic autoradiography can be used to identify the site of incorporation of a radiolabeled amino acid within the tissue and the specific cell and organelle in which it is utilized, or x-ray microanalysis can reveal the elemental composition of parts of cells or different cells within a tissue. Through the use of techniques like morphometry and stereology, morphologic studies can also be quantitative. To continue the example from above, the volume density ofthe radiolabeled organelte can he determined for a cell or tissue by means of selective sampling and prescribed methods of counting, thus allowing for quantitation and statistical analysis when comparing experimental data. Morphology should not have the limited connotation of providing purely descriptive information.The structure and composition of a tissue can provide insight into its function. As stated by Braverman and Braverman [2], "a major aim of biological work is to correlate structure with function in order to understand how tissues work." The goal in the present paper, as indicated by its title, is to support this statement as we review the use of morphologic approaches to understand skin hiology, selecting as an illustration the progress that has been made in understan...

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Flow Cytometry for Separation of Keratinocyte Subpopulations from the Viable Epidermis

Cited by 15 publications

References 21 publications

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies

Magnetic cell separation for purification of human oral keratinocytes: an effective method for functional studies without prior cell subcultivation

Biologic Structure and Function: Perspectives on Morphologic Approaches to the Study of the Granular Layer Keratinocyte.

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