A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose

Imaging is a powerful approach for studying protein expression and has the advantage over other methodologies in providing spatial information in situ at single cell level. Using immunofluorescence and confocal microscopy, detailed information of subcellular distribution of proteins can be obtained. While adherent cells of different tissue origin are relatively easy to prepare for imaging applications, non-adherent cells from hematopoietic origin, present a challenge due to their poor attachment to surfaces and subsequent loss of a substantial fraction of the cells. Still, these cell types represent an important part of the human proteome and express genes that are not expressed in adherent cell types. In the era of cell mapping efforts, overcoming the challenge with suspension cells for imaging applications would enable systematic profiling of hematopoietic cells. In this work, we successfully established an immunofluorescence protocol for preparation of suspension cell lines, peripheral blood mononucleated cells (PBMC) and human platelets on an adherent surface. The protocol is based on a multi-well plate format with automated sample preparation, allowing for robust high throughput imaging applications. In combination with confocal microscopy, the protocol enables systematic exploration of protein localization to all major subcellular structures.

Download Full-text

4D confocal microscopy method for drug localization in the skin

10.1117/12.889497 ◽

2011 ◽

Author(s):

Ulf Maeder ◽

Thorsten Bergmann ◽

Jan Michael Burg ◽

Sebastian Beer ◽

Peggy Schlupp ◽

...

Keyword(s):

Confocal Microscopy ◽

Microscopy Method ◽

Drug Localization

Download Full-text

Are They Really Stem Cells? Scrutinizing the Identity of Cells and the Quality of Reporting in the Use of Adipose Tissue-Derived Stem Cells

Stem Cells International ◽

10.1155/2016/2302430 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Ernesto Balolong ◽

Soojung Lee ◽

Judee Grace Nemeno ◽

Jeong Ik Lee

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Cellular Therapy ◽

Stromal Vascular Fraction ◽

Adherent Cells ◽

Quality Of Reporting ◽

Adequate Information ◽

Current Guidelines ◽

Multilineage Potential

There is an increasing concern that the term adipose tissue-derived stem cell (ASC) is inappropriately used to refer to the adipose stromal vascular fraction (SVF). To evaluate the accuracy and quality of reporting, 116 manuscripts on the application of ASC in humans and animals were examined based on the 2013 published International Federation for Adipose Therapeutics and Science (IFATS)/ International Society for Cellular Therapy (ISCT) joint statement and in reference to current guidelines for clinical trials and preclinical studies. It is disconcerting that 4 among the 47 papers or 8.51% (CI 2.37–20.38) surveyed after publication of IFATS/ISCT statement reported using ASCs but in fact they used unexpanded cells. 28/47 or 59.57% (CI 44.27–73.63) explicitly reported that adherent cells were used, 35/47 or 74.47% (CI 59.65–86.06) identified expression of surface markers, and 25/47 or 53.19% (CI 14.72–30.65) verified the multilineage potential of the cells. While there are a number of papers examined in this survey that were not able to provide adequate information on the characteristics of ASCs used with some erroneously referring to the SVF as stem cells, there are more room for improvement in the quality of reporting in the application of ASCs in humans and animals.

Download Full-text

Laser-scanning velocimetry: A confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae

BMC Biotechnology ◽

10.1186/1472-6750-7-40 ◽

2007 ◽

Vol 7 (1) ◽

pp. 40 ◽

Cited By ~ 40

Author(s):

Michael H Malone ◽

Noah Sciaky ◽

Lisa Stalheim ◽

Klaus M Hahn ◽

Elwood Linney ◽

...

Keyword(s):

Confocal Microscopy ◽

Laser Scanning ◽

Quantitative Measurement ◽

Zebrafish Embryos ◽

Cardiovascular Performance ◽

Microscopy Method

Download Full-text

Changes in endothelial actin cytoskeleton in venules with time after histamine treatment

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.269.5.h1528 ◽

1995 ◽

Vol 269 (5) ◽

pp. H1528-H1537 ◽

Cited By ~ 15

Author(s):

A. L. Baldwin ◽

G. Thurston

Keyword(s):

Endothelial Cells ◽

Confocal Microscopy ◽

Time Course ◽

Relative Size ◽

Fluorescein Isothiocyanate ◽

Adherent Cells ◽

Sprague Dawley ◽

Filamentous Actin ◽

Rhodamine Phalloidin ◽

Sprague Dawley Rats

In this study the time course of development and recovery of histamine-induced venular leaks was followed in conjunction with rearrangement of endothelial actin fibers. The microvasculature of a single mesenteric window of anesthetized Sprague-Dawley rats was perfused with buffered saline, with or without 10(-4) M histamine, for 3-30 min. Fluorescein isothiocyanate (FITC)-albumin was added for the last 3 min. The microvasculature was perfusion fixed, stained with rhodamine phalloidin (for filamentous actin), and viewed using confocal microscopy. The number and relative size of FITC-albumin leaks per venule length were measured. After 3 min of histamine application focal leaks appeared in some of the venules. Most focal leaks were accompanied by local breaks in the endothelial peripheral actin rim. Larger leaks were also present, accompanied by greater disruption of the venular endothelial peripheral actin rim, diffuse F-actin staining, and adherent platelets and leukocytes. Few central actin fibers were visible even in endothelial cells associated with large leaks. After 10-15 min of histamine exposure, larger leaks were more abundant but with fewer adherent cells. Central actin fibers in endothelial cells increased in number, peaking after 20 min of histamine, while the diffuse actin staining declined. Leak area per micrometer of venule peaked at 10-15 min, but the numbers of leaks per micrometer did not vary significantly from 3 to 30 min. These data suggest that the central fibers are not involved with the phase of increasing permeability, but they may play a role in the structural and functional recovery of endothelial cells perturbed by histamine.

Download Full-text

Исследование эффективноcти оптического просветления кожи растворами глицерина методом конфокальной микроспектроскопии комбинационного рассеяния света

Журнал технической физики ◽

10.21883/os.2020.06.49407.52-20 ◽

2020 ◽

Vol 128 (6) ◽

pp. 753

Author(s):

И.Ю. Янина ◽

И. Шлойзенер ◽

Ю. Ладеманн ◽

В.В. Тучин ◽

М.Е. Дарвин

Keyword(s):

Confocal Microscopy ◽

Stratum Corneum ◽

Dimethyl Sulfoxide ◽

Optical Parameters ◽

Distilled Water ◽

Optical Clearing ◽

Pig Skin ◽

Combination Scattering ◽

Microscopy Method ◽

Different Depths

The effect of glycerol solutions of various concentrations and exposure times on the effectiveness of optical clearing (OC) in intact pig skin was studied in the analysis of Raman spectra recorded at different depths. It was found that a solution of 80% glycerol and 20% dimethyl sulfoxide (DMSO) penetrated through the stratum corneum for 45 min. An increase in OC was achieved using mixtures of optical clearing agents (OCA) with DMSO and distilled water. So, the greatest OC efficiency was observed using a mixture of 60% glycerol and 40% water for 45 min (an increase of 3.4 times at a depth of 80 μm). Thus, the possibility of monitoring the optical parameters of the skin at a depth of about 80 μm using the confocal microscopy method of combination scattering.

Download Full-text

3-D organization of fibrinogen receptors using computer reconstruction and whole-mount microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102237 ◽

1988 ◽

Vol 46 ◽

pp. 30-31

Author(s):

E. T. O'Toole ◽

R. R. Hantgan ◽

J. C. Lewis

Keyword(s):

Whole Blood ◽

Colloidal Gold ◽

Blood Platelets ◽

Cell Contact ◽

Computer Assisted ◽

Adherent Cells ◽

Differential Centrifugation ◽

Computer Reconstruction ◽

Sds Page ◽

Whole Mount

Thrombocytes (TC), the avian equivalent of blood platelets, support hemostasis by aggregating at sites of injury. Studies in our lab suggested that fibrinogen (fib) is a requisite cofactor for TC aggregation but operates by an undefined mechanism. To study the interaction of fib with TC and to identify fib receptors on cells, fib was purified from pigeon plasma, conjugated to colloidal gold and used both to facilitate aggregation and as a receptor probe. Described is the application of computer assisted reconstruction and stereo whole mount microscopy to visualize the 3-D organization of fib receptors at sites of cell contact in TC aggregates and on adherent cells.Pigeon TC were obtained from citrated whole blood by differential centrifugation, washed with Ca++ free Hank's balanced salts containing 0.3% EDTA (pH 6.5) and resuspended in Ca++ free Hank's. Pigeon fib was isolated by precipitation with PEG-1000 and the purity assessed by SDS-PAGE. Fib was conjugated to 25nm colloidal gold by vortexing and the conjugates used as the ligand to identify fib receptors.

Download Full-text