scholarly journals A Sample Preparation Protocol for High Throughput Immunofluorescence of Suspension Cells on an Adherent Surface

2020 ◽  
Vol 68 (7) ◽  
pp. 473-489
Author(s):  
Anna Bäckström ◽  
Laura Kugel ◽  
Christian Gnann ◽  
Hao Xu ◽  
Joseph E. Aslan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Spatial Information ◽  
Protein Localization ◽  
Cell Types ◽  
Human Platelets ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Suspension Cells

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.

scholarly journals A sample preparation protocol for high throughput immunofluorescence of suspension cells

2020 ◽  
Author(s):  
Anna Bäckström ◽  
Laura Kugel ◽  
Christian Gnann ◽  
Hao Xu ◽  
Joseph E. Aslan ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Sample Preparation ◽  
High Throughput ◽  
Spatial Information ◽  
Protein Localization ◽  
Cell Types ◽  
Human Platelets ◽  
Adherent Cell ◽  
Suspension Cells ◽  
High Throughput Imaging

AbstractImaging 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, nonadherent 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 and peripheral blood mononucleated cells (PBMC) and human platelets. 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.

scholarly journals A method for incorporating macromolecules into adherent cells.

1984 ◽  
Vol 98 (4) ◽  
pp. 1556-1564 ◽  
Author(s):  
P L McNeil ◽  
R F Murphy ◽  
F Lanni ◽  
D L Taylor
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Cell Types ◽  
Stress Fibers ◽  
Human Neutrophils ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Simple Method ◽  
Fluorescent Molecules ◽  
Scrape Loading

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.

scholarly journals Tools for efficient analysis of neurons in a 3D reference atlas of whole mouse spinal cord

2021 ◽  
Author(s):  
Felix Fiederling ◽  
Luke A. Hammond ◽  
David Ng ◽  
Carol Mason ◽  
Jane Dodd
Keyword(s):  
Spinal Cord ◽  
Sample Preparation ◽  
High Throughput ◽  
Adult Mouse ◽  
Spatial Information ◽  
Spinal Neurons ◽  
Mouse Spinal Cord ◽  
Single Block ◽  
And Function

Spinal neurons are highly heterogeneous in location, transcriptional identity and function. To understand their contributions to sensorimotor circuits, it is essential to map the positions of identified subsets of neurons in relation to others throughout the spinal cord (SC), but we lack tools for whole SC sample preparation, imaging and in toto analysis. To overcome this problem, we have (1) designed scaffolds (SpineRacks) that facilitate efficient and ordered cryo-sectioning of the entire SC in a single block, (2) constructed a 3D reference atlas of adult mouse SC and (3) developed software (SpinalJ) to register images of sections and for standardized analysis of cells and projections in atlas space. We have verified mapping accuracies for known neurons and demonstrated the usefulness of this platform to reveal unknown neuronal distributions. Together, these tools provide high-throughput analyses of whole mouse SC and enable direct comparison of 3D spatial information between animals and studies.

scholarly journals Erythropoietic repopulating ability of stem cells from long-term marrow culture

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1021-1025 ◽  
Author(s):  
DE Harrison ◽  
CP Lerner ◽  
E Spooncer
Keyword(s):  
Stem Cells ◽  
Cell Types ◽  
Adherent Cells ◽  
Hemopoietic Precursors ◽  
Suspension Cells ◽  
Direct Measurements ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Marrow Cells

Abstract Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU- S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.

High-throughput functional profiling of single adherent cells via hydrogel drop-screen

Lab on a Chip ◽  
2021 ◽  
Vol 21 (4) ◽  
pp. 764-774
Author(s):  
Ming Wang ◽  
Mui Hoon Nai ◽  
Ruby Yun-Ju Huang ◽  
Hwa Liang Leo ◽  
Chwee Teck Lim ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Functional Profiling

A hydrogel drop-screen device was developed to rapidly measure large-scale single-adherent cell morphologies and multiple protease secretions on gelatin particles with a throughput ∼100 cells per second for phenotype profiling.

scholarly journals Erythropoietic repopulating ability of stem cells from long-term marrow culture

Blood ◽  
1987 ◽  
Vol 69 (4) ◽  
pp. 1021-1025 ◽  
Author(s):  
DE Harrison ◽  
CP Lerner ◽  
E Spooncer
Keyword(s):  
Stem Cells ◽  
Cell Types ◽  
Adherent Cells ◽  
Hemopoietic Precursors ◽  
Suspension Cells ◽  
Direct Measurements ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Marrow Cells

Hemopoietic precursors are heterogeneous with respect to their capacity for self-renewal and long-term repopulating ability. Bone marrow cultures produce a variety of precursors over many weeks, including CFU- S; however, it is important to determine whether these populations retain the functional ability shown by fresh marrow. The most primitive precursor or stem cells have the most long-term repopulating ability. We here describe direct measurements of this ability in cells from marrow cultures by using competitive repopulation assays. Cultured adherent cells repeatedly showed less capacity than fresh marrow cells to repopulate erythropoiesis in irradiated recipients, whereas cultured suspension cells consistently had less capacity than adherent cells. Concentrations of macroscopic CFU-S measured at nine or 12 days were similar in cultured adherent and suspension cells and generally lower than those in fresh marrow. In every experiment, the long-term repopulating ability of the marrow cells used was substantially reduced after transfer into tissue culture. Thus, primitive stem cells may not proliferate in such cultures despite extensive production of CFU-S and more differentiated cell types.

scholarly journals Cell Adhesion Promotes Ebola Virus Envelope Glycoprotein-Mediated Binding and Infection

2008 ◽  
Vol 82 (14) ◽  
pp. 7238-7242 ◽  
Author(s):  
Derek Dube ◽  
Kathryn L. Schornberg ◽  
Tzanko S. Stantchev ◽  
Matthew I. Bonaparte ◽  
Sue E. Delos ◽  
...  
Keyword(s):  
Envelope Glycoprotein ◽  
Ebola Virus ◽  
Cell Spreading ◽  
Cell Types ◽  
Pseudotyped Virus ◽  
Adherent Cell ◽  
Adherent Cells ◽  
Receptor Binding Domain ◽  
Virus Envelope ◽  
Nonadherent Cells

ABSTRACT Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

scholarly journals PC12 Cell Line: Cell Types, Coating of Culture Vessels, Differentiation and Other Culture Conditions

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 958 ◽  
Author(s):  
Benita Wiatrak ◽  
Adriana Kubis-Kubiak ◽  
Agnieszka Piwowar ◽  
Ewa Barg
Keyword(s):  
Cell Line ◽  
Pc12 Cells ◽  
Pc12 Cell ◽  
Cell Types ◽  
Culture Conditions ◽  
Adherent Cells ◽  
Suspension Cells ◽  
Pc12 Cell Line ◽  
Collagen Coating ◽  
Coated Surfaces

The PC12 cell line is one of the most commonly used in neuroscience research, including studies on neurotoxicity, neuroprotection, neurosecretion, neuroinflammation, and synaptogenesis. Two types of this line are available in the ATCC collection: traditional PC12 cells grown in suspension and well-attached adherent phenotype. PC12 cells grown in suspension tend to aggregate and adhere poorly to non-coated surfaces. Therefore, it is necessary to modify the surface of culture vessels. This paper aims to characterise the use of two distinct variants of PC12 cells as well as describe their differentiation and neuronal outgrowth with diverse NGF concentrations (rat or human origin) on various surfaces. In our study, we evaluated cell morphology, neurite length, density and outgrowth (measured spectrofluorimetrically), and expression of neuronal biomarkers (doublecortin and NeuN). We found that the collagen coating was the most versatile method of surface modification for both cell lines. For adherent cells, the coating was definitely less important, and the poly-d-lysine surface was as good as collagen. We also demonstrated that the concentration of NGF is of great importance for the degree of differentiation of cells. For suspension cells, we achieved the best neuronal characteristics (length and density of neurites) after 14 days of incubation with 100 ng/mL NGF (change every 48 h), while for adherent cells after 3–5 days, after which they began to proliferate. In the PC12 cell line, doublecortin (DCX) expression in the cytoplasm and NeuN in the cell nucleus were found. In turn, in the PC12 Adh line, DCX was not expressed, and NeuN expression was located in the entire cell (both in the nucleus and cytoplasm). Only the traditional PC12 line grown in suspension after differentiation with NGF should be used for neurobiological studies, especially until the role of the NeuN protein, whose expression has also been noted in the cytoplasm of adherent cells, is well understood.

scholarly journals Stem Cell Heterogeneity of Mononucleated Cells from Murine Peripheral Blood: Molecular Analysis

2011 ◽  
Vol 11 ◽  
pp. 2150-2159 ◽  
Author(s):  
Muhammad Dain Yazid ◽  
Shahrul Hisham Zainal Ariffin ◽  
Sahidan Senafi ◽  
Zaidah Zainal Ariffin ◽  
Rohaya Megat Abdul Wahab
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Cell Types ◽  
Complete Medium ◽  
Adherent Cells ◽  
Suspension Cells ◽  
Isolated Cells ◽  
Hematopoietic Stem ◽  
Culture Time ◽  
Different Cell Types

The main purpose of this paper was to determine the heterogeneity of primary isolated mononucleated cells that originated from the peripheral blood system by observing molecular markers. The isolated cells were cultured in complete medium for 4 to 7 days prior to the separation of different cell types, that is, adherent and suspension. Following a total culture time of 14 days, adherent cells activated theCd105gene while suspension cells activated theSca-1gene. Both progenitor markers,Cbfa-1andOstf-1, were inactivated in both suspension and adherent cells after 14-day culture compared to cells cultured 3 days in designated differentiation medium. In conclusion, molecular analyses showed that primary mononucleated cells are heterogeneous, consisting of hematopoietic stem cells (suspension) and mesenchymal stem cells (adherent) while both cells contained no progenitor cells.

High-Throughput, Site-Specific Sample Prep of Ultra-Thin TEM Lamella for Process Metrology and Failure Analysis

2012 ◽  
Author(s):  
Roger Alvis ◽  
Jeff Blackwood ◽  
Sang-Hoon Lee ◽  
Matthew Bray
Keyword(s):  
Sample Preparation ◽  
Failure Analysis ◽  
Process Monitoring ◽  
High Throughput ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Memory Devices ◽  
Site Specific ◽  
Critical Dimensions ◽  
Tem Lamella

Abstract Semiconductor devices with critical dimensions less than 20nm are now being manufactured in volume. A challenge facing the failure analysis and process-monitoring community is two-fold. The first challenge of TEM sample prep of such small devices is that the basic need to end-point on a feature-of-interest pushes the imaging limit of the instrument being used to prepare the lamella. The second challenge posed by advanced devices is to prepare an artifact-free lamella from non-planar devices such as finFETs as well as from structures incorporating ‘non-traditional’ materials. These challenges are presently overcome in many advanced logic and memory devices in the focused ion beam-based TEM sample preparation processes by inverting the specimen prior to thinning to electron transparency. This paper reports a highthroughput method for the routine preparation of artifact-free TEM lamella of 20nm thickness, or less.

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