scholarly journals Magnetic Cell Sorting for In Vivo and In Vitro Astrocyte, Neuron, and Microglia Analysis

2019 ◽  
Vol 88 (1) ◽  
Author(s):  
Leanne M. Holt ◽  
S. Tristan Stoyanof ◽  
Michelle L. Olsen
Keyword(s):  
Cell Sorting ◽  
Magnetic Cell Sorting

scholarly journals Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3469-3473 ◽  
Author(s):  
JA Christian ◽  
AH Rebar ◽  
GD Boon ◽  
PS Low
Keyword(s):  
Life Span ◽  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Magnetic Cell Sorting ◽  
Autologous Plasma ◽  
Low Degree ◽  
La Jolla ◽  
Immunoglobulin Binding

Abstract We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.

scholarly journals Senescence of canine biotinylated erythrocytes: increased autologous immunoglobulin binding occurs on erythrocytes aged in vivo for 104 to 110 days

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3469-3473 ◽  
Author(s):  
JA Christian ◽  
AH Rebar ◽  
GD Boon ◽  
PS Low
Keyword(s):  
Life Span ◽  
Cell Sorting ◽  
Gradient Centrifugation ◽  
Magnetic Cell Sorting ◽  
Autologous Plasma ◽  
Low Degree ◽  
La Jolla ◽  
Immunoglobulin Binding

We have evaluated senescence related changes in canine red blood cells (RBCs) using the biotinylation system, where RBCs are labeled in vivo with biotin at the beginning of their life span, and retrieved from circulation on immobilized avidin at the end of their life span. This approach avoids the controversial use of density gradient centrifugation to collect presumably old RBCs. Furthermore, the dog is an appropriate model for human RBC senescence because it has a low degree of random RBC loss and a similarly long RBC life span (approximately 110 days). Two dogs had 97% to 100% of their circulating RBCs biotinylated by infusion of N-hydroxysuccinimido biotin (Clontech, Palo Alto, CA; Calbiochem, La Jolla, CA) dissolved in dimethyl sulfoxide. At postbiotinylation days 104 and 107 for one dog and day 110 for the other dog, biotinylated RBCs were isolated by magnetic cell sorting and analyzed for the presence of autologous IgG using 125I- labeled sheep-antidog IgG (SAD IgG). On all 3 days, there were at least three times more SAD IgG molecules per RBC on senescent biotinylated RBCs than on control (unfractionated) RBCs (day 104: 11,677 v 3,399; day 107: 6,710 v 2,115; day 110: 6,042 v 1,838 molecules of SAD IgG per senescent v control RBC). Furthermore, it is unlikely that an immune response to the conjugated biotin had been elicited, because fresh in vitro biotinylated RBCs that were incubated in autologous plasma (taken after exposure to circulating biotinylated RBCs for 113 days) and then exposed to the SAD IgG showed no increase in antibody binding over control (non-biotinylated) RBCs (1,431 v 1,378 cpm/10(8) biotinylated v control RBCs; P > .20). These results suggest that senescence of canine biotinylated RBCs is characterized by binding of autologous IgG and that antibiotin antibodies do not contribute to this process.

scholarly journals Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

2020 ◽  
Vol 22 (1) ◽  
pp. 314
Author(s):  
Maria D. Dmitrieva ◽  
Anna A. Voitova ◽  
Maya A. Dymova ◽  
Vladimir A. Richter ◽  
Elena V. Kuligina
Keyword(s):  
Phage Display ◽  
Cell Sorting ◽  
Targeted Delivery ◽  
Tumor Targeting ◽  
Search Strategy ◽  
Tumor Therapy ◽  
Therapeutic Agents ◽  
Phage Libraries

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging

2007 ◽  
Vol 293 (5) ◽  
pp. C1698-C1708 ◽  
Author(s):  
Y. Jeffrey Wu ◽  
Leslie L. Muldoon ◽  
Csanad Varallyay ◽  
Sheila Markwardt ◽  
Richard E. Jones ◽  
...  
Keyword(s):  
Cell Sorting ◽  
B Lymphocyte ◽  
Protamine Sulfate ◽  
Mononuclear Leukocytes ◽  
Intracerebral Injection ◽  
Sulfate Complex ◽  
Human Dose ◽  
The Brain

Cellular labeling with ferumoxides (Feridex IV) superparamagnetic iron oxide nanoparticles can be used to monitor cells in vivo by MRI. The objective of this study was to use histology and MRI to evaluate an in vivo, as opposed to in vitro, technique for labeling of mononuclear leukocytes as a means of tracking inflammatory processes in the brain. Long-Evans rats were intravenously injected with 20 mg/kg ferumoxides, ferumoxtran-10, or ferumoxytol with or without protamine sulfate. Leukocytes and splenocytes were evaluated by cell sorting and iron histochemistry or were implanted into the brain for MRI. Injection of ferumoxides/protamine sulfate complex IV resulted in iron labeling of leukocytes (ranging from 7.4 ± 0.5% to 12.5 ± 0.9% with average 9.2 ± 0.8%) compared with ferumoxides (ranging from 3.9 ± 0.4% to 6.3 ± 0.5% with average 5.0 ± 0.5%) or protamine sulfate alone (ranging from 0% to 0.9 ± 0.7% with average 0.3 ± 0.3%). Cell sorting analysis indicated that iron-labeled cells were enriched for cell types positive for the myelomonocytic marker (CD11b/c) and the B lymphocyte marker (CD45RA) and depleted in the T cell marker (CD3). Neither ferumoxtran-10 nor ferumoxytol with protamine sulfate labeled leukocytes. In vivo ferumoxides/protamine sulfate-loaded leukocytes and splenocytes were detected by MRI after intracerebral injection. Ferumoxides/protamine complex labeled CD45RA-positive and CD11b/c-positive leukocytes in vivo without immediate toxicity. The dose of feumoxides in this report is much higher than the approved human dose, so additional animal studies are required before this approach could be translated to the clinic. These results might provide useful information for monitoring leukocyte trafficking into the brain.

Analysis of the hematopoietic potential of muscle-derived cells in mice

Blood ◽  
2002 ◽  
Vol 100 (2) ◽  
pp. 721-723 ◽  
Author(s):  
Hartmut Geiger ◽  
Jarrod M. True ◽  
Barry Grimes ◽  
Elizabeth J. Carroll ◽  
Roger A. Fleischman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Muscle Tissue ◽  
Cell Sorting ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Muscle Stem Cells ◽  
Antibody Staining ◽  
Stem And Progenitor Cells

Abstract Cells in murine muscle have been reported to differentiate into hematopoietic stem and progenitor cells and thus repopulate the hematopoietic system of an irradiated animal. This activity was attributed to muscle stem cells. We used an in vitro and in vivo approach to identify the hematopoietic repopulating activity found in muscle tissue of mice by antibody staining and cell sorting. We confirmed existence of a hematopoietic repopulating cell in muscle tissue, but the data strongly suggest that repopulation is due not to muscle stem cells but to hematopoietic cells present in muscle tissue. Unexpectedly, the blood-forming cells were enriched in muscle relative to their frequency in peripheral blood.

scholarly journals Epiblast cells that express MyoD recruit pluripotent cells to the skeletal muscle lineage

2004 ◽  
Vol 164 (5) ◽  
pp. 739-746 ◽  
Author(s):  
Jacquelyn Gerhart ◽  
Christine Neely ◽  
Benjamin Stewart ◽  
Jordanna Perlman ◽  
David Beckmann ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Sorting ◽  
Embryonic Stem ◽  
Bmp Signaling ◽  
Soluble Form ◽  
Skeletal Myogenesis ◽  
Magnetic Cell Sorting ◽  
Pluripotent Cells ◽  
Morphogenetic Protein

Embryonic stem cells are derived from the epiblast. A subpopulation of epiblast cells expresses MyoD mRNA and the G8 antigen in vivo. G8 positive (G8pos) and G8 negative (G8neg) populations were isolated by magnetic cell sorting. Nearly all G8pos cells switched from E- to N-cadherin and differentiated into skeletal muscle in culture. G8neg cells were impaired in their ability to switch cadherins and few formed skeletal muscle. Medium conditioned by G8pos cells stimulated skeletal myogenesis and N-cadherin synthesis in G8neg cultures. The effect of conditioned medium from G8pos cultures was inhibited by bone morphogenetic protein (BMP) 4. Treatment of G8neg cells with a soluble form of the BMP receptor-IA or Noggin promoted N-cadherin synthesis and skeletal myogenesis. These results demonstrate that MyoD-positive epiblast cells recruit pluripotent cells to the skeletal muscle lineage. The mechanism of recruitment involves blocking the BMP signaling pathway.

scholarly journals Osteal Macrophages and Megakaryocytes Increase Adipogenesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Nikhil Tewari ◽  
Deepa Kanagasabapathy ◽  
Rachel J. Blosser ◽  
Edward F. Srour ◽  
Angela Bruzzaniti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Sorting ◽  
Fetal Liver ◽  
Fold Increase ◽  
Wild Type ◽  
Bone Marrow Adipose Tissue ◽  
New Treatments

Bone marrow adipose tissue (MAT) increases with aging and contributes to low bone density and skeletal fractures. However, the cells and factors within the bone marrow (BM) that regulate adipogenesis remain poorly understood. In the current study, we examined the role of osteal macrophages (OMs) and megakaryocytes (MKs) on the regulation of adipogenesis. We cultured murine osteoblasts/osteoblast progenitors (OBs from hereon) derived from neonatal calvarial cells (CCs, a combination of OBs and OMs) or OBs isolated by fluorescence activated cell sorting (FACS) in the presence or absence of fetal liver derived murine MK. The cells underwent induced adipogenesis for 5-7 days by supplementation of media with insulin, indomethacin, and dexamethasone, and then the number of adipocytes was quantified.   We found that co-culturing MKs and OMs with OBs results in up to a 7.8-fold and 11.7-fold increase in adipocytes, respectively. We also elucidated that thrombopoietin (TPO), the major growth factor for MKs, inhibits adipogenesis in both OBs and CCs by approximately 60%. Similarly, we found that CCs and OBs derived from mice deficient in the TPO receptor, Mpl, had approximately 30% more adipocytes than their wild-type (WT) counterparts. Finally, in vitro findings were corroborated in vivo through quantification of MKs and adipocytes in mice in which MK number was elevated or reduced. Mice with significantly higher numbers of BM-residing MKs also had significantly higher numbers of BM-residing adipocytes. Because there is typically an inverse relationship between adipogenesis and osteogenesis, understanding ways to inhibit adipogenesis could lead to an increase in OB number and bone formation, which in turn could lead to new treatments for bone loss diseases such as osteoporosis.

138 IN VITRO CULTURE OF CD9-EXPRESSING CELLS ENRICHED BY MAGNETIC CELL SORTING FROM TESTES OF CRYPTORCHID ADULT AND PUP IN MICE

2006 ◽  
Vol 18 (2) ◽  
pp. 177
Author(s):  
T. Mitani ◽  
Y. Tanaka ◽  
Y. Ozaki ◽  
K. Saeki ◽  
K. Kato ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Surface ◽  
Germ Cells ◽  
Cell Sorting ◽  
Es Cells ◽  
Immunohistochemical Analysis ◽  
Cell Suspensions ◽  
Magnetic Cell Sorting ◽  
Fcm Analysis

Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation techniques have made possible the functional analysis of germline stem cells (GS cells). The GS cells are downstream of the stem cells such as ES cells and embryonic germ cells (EG cells), which are derived from primordial germ cells (PGCs). Therefore, GS cells are expected to be useful in the production of genetically modified animals. In this study, we examined the enrichment and cultivation of mouse GS cells by magnetic cell sorting (MACS). Testicular cell suspensions were collected from C57BL/6J cryptorchid adult testes at 2 to 3 months after surgery and ICR pup (6 to 8 dpp) testes. They were digested by 0.1% collagenase followed by 0.25% trypsin with gentle shaking. Dissociated cell suspensions were filtrated through a glass-wool column followed by a Falcon cell strainer (40-�m mesh). They were then treated with biotin-conjugated anti-mouse CD9 antibody, whose antigen, CD9, is localized on the GS cell surface, followed by the streptavidin-microbeads treatment. The cell suspension was passed through a MACS-separation column. In Experiment I, MACS-treated fractions were analyzed by flow cytometry (FCM) on the rates of recovery and enrichment and their cellular characteristics. In Experiment II, CD9-positive (CD9+) cells were cultured on gelatin-coated MultiDish (176740, Nunc) with 4-5 � 105 cells/well in StemPro34-SFM supplemented with 1% fetal bovine serum, leukemia inhibitory factor, GDNF, bFGF, EGF, insulin, transferrin, putrescine, MEM vitamin solution, MEM-NEAA and some other reagents at 32�C or 37�C under 5% CO2 in air. They were examined for their proliferation and cytological changes such as CD9, �6-integrin and Oct-1 expression by immunohistochemistry. In Experiment I, MACS selection effectively enriched CD9+ cells from mouse testes. However, FCM analysis revealed that the CD9-negative (CD9-) cells partially remained in MACS-selected fraction from cryptorchid adult testes. In contrast, the CD9+ subpopulation could be successfully separated from CD9- subpopulation from pup testes. Therefore CD9+ subpopulation from pup testes was used for the following cultivation. In Experiment II, the cells proliferated in the first few days in suspension. Then they attached to the dish and formed colonies after 5 days or 3 days of culture at 32�C or 37�C, respectively. Immunohistochemical analysis showed that the cells maintained the expression of CD9 for at least 14 days, but their expression of �6-integrin gradually diminished. It was demonstrated by immunohistochemistry and FCM analysis that the cells in colonies expressed Oct-1, and its expression level was stronger in culture at 37�C than at 32�C. These findings indicate that the CD9+ cells collected from mouse pup testes have stem cell properties. This work was supported by the Wakayama Prefecture Collaboration of Regional Entities for the Advanced Technological Excellence, JST; by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan; and by a Grant-in Aid for Scientific Research from the Japan Society for the Promotion of Science.

scholarly journals Forces driving cell sorting in Hydra

2017 ◽  
Author(s):  
Olivier Cochet-Escartin ◽  
Tiffany T. Locke ◽  
Winnie H. Shi ◽  
Robert E. Steele ◽  
Eva-Maria S. Collins
Keyword(s):  
Cell Sorting ◽  
Single Cells ◽  
Cell Types ◽  
Biological Organization ◽  
3 Dimensional ◽  
Physical Mechanisms ◽  
Summary Statement ◽  
Endodermal Cells

AbstractCell sorting, whereby a heterogeneous cell mixture organizes into distinct tissues, is a fundamental patterning process in development. So far, most studies of cell sorting have relied either on 2-dimensional cellular aggregates, in vitro situations that do not have a direct counterpart in vivo, or were focused on the properties of single cells. Here, we report the first multiscale experimental study on 3-dimensional regenerating Hydra aggregates, capable of reforming a full animal. By quantifying the kinematics of single cell and whole aggregate behaviors, we show that no differences in cell motility exist among cell types and that sorting dynamics follow a power law. Moreover, we measure the physical properties of separated tissues and determine their viscosities and surface tensions. Based on our experimental results and numerical simulations, we conclude that tissue interfacial tensions are sufficient to explain Hydra cell sorting. Doing so, we illustrate D’Arcy Thompson’s central idea that biological organization can be understood through physical principles, an idea which is currently re-shaping the field of developmental biology.Summary statementHydra regenerates after dissociation into single cells. We show how physical mechanisms can explain the first step of regeneration, whereby ectodermal and endodermal cells sort out to form distinct tissue layers.

scholarly journals Identification of a New Virulence Factor, BvfA, in Brucella suis

2005 ◽  
Vol 73 (9) ◽  
pp. 5524-5529 ◽  
Author(s):  
Jean-Philippe Lavigne ◽  
Gilles Patey ◽  
Felix J. Sangari ◽  
Gisèle Bourg ◽  
Michel Ramuz ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Cell Sorting ◽  
Fluorescent Protein ◽  
Intracellular Replication ◽  
Knockout Mutant ◽  
Macrophage Infection ◽  
Brucella Suis ◽  
Green Fluorescent

ABSTRACTWe report the identification of BvfA (forBrucellavirulence factor A), a small periplasmic protein unique to the genusBrucella, which is essential for the virulence ofBrucella suis. A BvfA knockout mutant was highly attenuated both in in vitro macrophage infection assays and in vivo in the murine model of brucellosis. Fluorescence-activated cell sorting analysis with green fluorescent protein fusions showed that the expression ofbvfAis induced within macrophages by phagosome acidification and coregulated with theB. suis virBoperon, suggesting that it too may play a role in the establishment of the intracellular replication niche.

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