Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications

2018 ◽  
Vol 73 (3-4) ◽  
pp. 95-105 ◽  
Author(s):  
Mohamed K. Al-Essa ◽  
Susanne Melzer ◽  
Attila Tarnok ◽  
Kamal A. Hadidi ◽  
Mohammed El-Khateeb
Keyword(s):  
Succinic Anhydride ◽  
Fluorescent Dyes ◽  
Fluorescein Isothiocyanate ◽  
Fold Increase ◽  
Fluorescent Dye ◽  
Cooh Group ◽  
Vitro Assay ◽  
Bioanalytical Applications ◽  
Ethanesulfonic Acid

AbstractThis study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4′,6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

scholarly journals Phagocytosis of phenylhydrazine oxidized and G-6-PD-deficient red blood cells: the role of cell-bound immunoglobulins

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1818-1825 ◽  
Author(s):  
S Horn ◽  
N Bashan ◽  
J Gopas
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Fold Increase ◽  
Fc Receptor ◽  
Autologous Serum ◽  
Murine Macrophages

Abstract In this study, the role of Igs in the recognition and removal of oxidatively damaged human red blood cells (RBCs) was investigated. Phagocytosis of normal RBCs exposed to the oxidative hemolytic agent phenylhydrazine (Phz) and of glucose-6-phosphate dehydrogenase (G6PD)- deficient RBCs by murine macrophages was examined. A 40-fold increase in phagocytosis of RBCs treated with 3 mmol/L Phz was obtained both in the absence and presence of autologous serum, indicating that binding of autologous antibodies to the oxidized cells is not essential for phagocytosis. Yet, a basal number of IgG molecules was found to be present on the RBCs, as determined both by binding of 125I protein A and fluorescein isothiocyanate-antihuman Ig to the cells. Macrophage Fc receptors were found to be involved in the recognition of the RBCs, because phagocytosis was partially inhibited by incubating macrophages with bovine serum albumin (BSA) anti-BSA complexes, aIg (aggregated Igs), and anti-Fc receptor II monoclonal antibodies. Galactose/mannose inhibited phagocytosis of oxidized RBCs additively to aIg. Because phagocytosis was decreased when Phz-RBCs were incubated with F(ab')2 fragments of antihuman antibodies, it is suggested that the basal amount of Igs bound to the cells plays a role in the recognition of Phz- RBCs. G6PD-deficient RBCs were recognized and phagocytosed by murine macrophages without preexposure to oxidants in vitro (mean of 19 RBCs/100 macrophages). This phagocytosis was not affected by the addition of serum and was inhibited by incubating macrophages with galactose/mannose and the various Fc receptor blockers. A positive correlation between hemoglobin content and the number of cell-bound Igs to each patient erythrocytes was found. These results support the involvement of both an Fc and a lectin-like macrophage receptor in the recognition and phagocytosis of Phz-oxidized and G6PD-deficient RBCs and suggest opsonization as a possible physiologic process for the removal of severe damaged RBCs.

Leukotriene B4-induced neutrophil-mediated endothelial leakage in vitro and in vivo

1991 ◽  
Vol 71 (4) ◽  
pp. 1322-1330 ◽  
Author(s):  
S. Rosengren ◽  
A. M. Olofsson ◽  
U. H. von Andrian ◽  
E. Lundgren-Akerlund ◽  
K. E. Arfors
Keyword(s):  
Umbilical Vein ◽  
Neutrophil Migration ◽  
Fluorescein Isothiocyanate ◽  
Leukotriene B4 ◽  
Vascular Leakage ◽  
Neutrophil Granulocytes ◽  
Vitro Assay ◽  
Umbilical Vein Endothelial Cells

The vascular leakage of macromolecules seen in several models after application of leukotriene B4 (LTB4) is mediated by neutrophil granulocytes. We describe here an in vitro assay for this event. Human umbilical vein endothelial cells were grown on polycarbonate filters separating luminal and abluminal compartments of fluid. Both clearance rate of fluorescein isothiocyanate albumin and neutrophil migration through the endothelial monolayer were increased when LTB4 (10–100 nM) was added to the abluminal compartment. However, if LTB4 was instead added to the luminal compartments together with the neutrophils, no migration or change in clearance could be detected. These findings were confirmed in vivo in the cheek pouches of anesthetized hamsters, where extravascular application of LTB4 induced intravascular adhesion of neutrophils, accompanied by neutrophil-dependent vascular leakage. On the other hand, intravascular deposition of LTB4 with micropipettes induced adhesion of leukocytes but no leakage. In conclusion, the presence of neutrophils adhering to endothelium does not necessarily imply the development of neutrophil-mediated vascular leakage. Instead, the leakage appears connected to the process of neutrophil chemotaxis.

Influence of Cl- on pH(i) in oxynticopeptic cells of in vitro frog gastric mucosa

1990 ◽  
Vol 258 (5) ◽  
pp. G815-G824 ◽  
Author(s):  
A. Yanaka ◽  
K. J. Carter ◽  
H. H. Lee ◽  
W. Silen
Keyword(s):  
Gastric Mucosa ◽  
Intracellular Ph ◽  
Rana Catesbeiana ◽  
Basolateral Membrane ◽  
Fluorescent Dye ◽  
Disulfonic Acid ◽  
Fundic Mucosa ◽  
Ethanesulfonic Acid ◽  
Oxynticopeptic Cells

The effect of Cl- on intracellular pH (pH(i)) was studied using sheets of frog (Rana catesbeiana) fundic mucosa in which oxynticopeptic cells were selectively loaded with the acetomethoxy ester form of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). Before the measurement of pH(i), tissues were exposed to either 10(-5) M forskolin in the serosal solution (stimulated tissues) or 3 x 10(-4) omeprazole in the serosal solution (inhibited tissues). In HCO3- and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffers, pH(i) increased significantly after removal of Cl- from serosal and luminal solution, both in stimulated and inhibited tissues. The presence of Cl- in the luminal solution prevented this rise in pHi, an effect abolished by serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 3 x 10(-4) M) but not by serosal amiloride (10(-3)M). In the presence of serosal Cl-, pH(i) increased after exposure to serosal DIDS, more prominently in the stimulated than in the inhibited tissues. These results confirm the presence of a Cl(-)-HCO3-exchanger in the basolateral membrane of oxynticopeptic cells in intact sheets of mucosa and suggest that luminal Cl- contributes to the regulation of pH(i) in oxynticopeptic cells.

Abstract 580: Acidic Domain of ApoB-100 and Other Short Negatively Charged Peptides Anchored to Lipoproteins Cause Hypertriglyceridemia in Mice

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Denis O Sviridov ◽  
Puja Terse ◽  
Anna Wolska ◽  
Scott M Gordon ◽  
Alan T Remaley
Keyword(s):  
Amino Acid ◽  
Fold Increase ◽  
Alpha Tocopherol ◽  
Binding Motif ◽  
Vitro Assay ◽  
Acidic Domain ◽  
Ldl Particles ◽  
Control Peptide ◽  
Charged Peptides

Human Apolipoprotein B-100 (ApoB-100) is the main protein of VLDL and LDL particles in plasma. It is a large 4536 amino acid protein with various structural domains, such as the LDL-receptor binding motif and multiple proteoglycan binding sites, all of which have a high positive charge. A search of the primary amino acid sequence of ApoB-100 revealed the presence of the following acidic domain: DMDEDDD (AA 3985-3991). We hypothesized that this motif could affect the interaction of ApoB-containing lipoproteins with liver and or possibly GPIHBP1, which also contains an acidic domain that interacts with the positive charged motifs on VLDL. We synthesized a fluorescent tagged DMDEDDD peptide and conjugated it at the N-terminus with two molecules of alpha-tocopherol to serve as an anchor to lipoprotein particles. After adding the peptide to human plasma, it was found to readily bind to all lipoproteins, as determined by FPLC analysis. VLDL incubated with the peptide showed an increased negative charge and lost its ability to bind to heparin as determined by chromatography on a HiTrap TM heparin column. The peptide injected in LDLr-KO mice (30 mg/kg) caused a rapid 3-fold increase in plasma triglycerides within 1 h and the triglyceride elevation persisted for at least 24 h. A similarly designed negative charged peptide attached to tocopherol (EEEEEEEE) also raised triglycerides in mice but a neutral charged control peptide (KEKEKEKE) did not. To understand the mechanism, we tested the above peptides for their ability to inhibit LPL, using an in vitro assay with intralipid as a substrate and found that negative charged peptides inhibited lipolysis (IC 50= 50 uM), but the neutral control peptide did not. We are now investigating other potential mechanisms for how acidic peptides may cause hypertriglyceridemia. In summary, a peptide based on an acidic domain on ApoB-100 appears to cause hypertriglyceridemia in mice by inhibition of LPL and possibly by interfering with proteoglycan binding of lipoproteins. These results may be relevant in understanding VLDL metabolism and for the design of therapeutic apolipoprotein mimetic peptides.

Abstract 293: Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Vasculogenesis.

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Lucinda Low ◽  
Christine Cheung ◽  
Martin R Bennett ◽  
Sanjay Sinha
Keyword(s):  
Network Formation ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Confocal Imaging ◽  
Paraxial Mesoderm ◽  
Hesc Line ◽  
Vitro Assay ◽  
Matrigel Assay ◽  
Network Area

Lineage-tracking studies in avian and mouse embryos have revealed that SMCs in different regions of the vasculature are derived from different embryological origins. We have developed an in vitro protocol to differentiate human embryonic stem cells (hESCs) into SMCs through different embryonic lineages, namely neuroectoderm, lateral plate mesoderm (LM) and paraxial mesoderm. As mural cells are thought to augment the formation of functional blood vessels during revascularisation, this study aims to determine whether embryologically-distinct SMCs differ in their ability to support vasculogenesis. We assessed the hypothesis that LM-SMCs are superior in supporting endothelial network formation in vitro. Using an mStrawberry-expressing hESC line, we derived the three origin-specific SMCs and co-cultured them with GFP-expressing HUVECs in a 3D in vitro matrigel assay. Endothelial network formation was assessed using real-time confocal imaging. Quantitative analysis revealed that LM-SMCs alone had a supportive effect on network formation and survival, with an increase in HUVEC network area after 4 days (3.23-fold increase ± 1.04-fold vs HUVECs alone; p<0.05; n=3) and 8 days (6.38-fold increase ± 0.39-fold vs HUVECs alone; p<0.001; n=3). In addition, LM-SMCs appear to facilitate more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. To identify whether the LM-SMC-specific influence on network formation and survival was a paracrine effect, the three SMC types were tested in a paracrine 3D in vitro assay, where they shared media with HUVECs seeded in an adjacent well. Preliminary results reveal that the supportive effect of LM-SMCs is, at least partly, paracrine, with an increase in HUVEC network area after 4 days (7.08-fold increase vs HUVECs alone; n=1) and 8 days (21.39-fold increase vs HUVECs alone; n=1). Currently, we are exploring possible soluble factors that may be responsible and investigating this effect in an in vivo matrigel assay. In conclusion, the embryological origin of SMCs influences their functional ability to support vasculogenesis. This research will provide insight into which SMC type will be most effective in future revascularisation therapy.

scholarly journals Phagocytosis of phenylhydrazine oxidized and G-6-PD-deficient red blood cells: the role of cell-bound immunoglobulins

Blood ◽  
1991 ◽  
Vol 78 (7) ◽  
pp. 1818-1825
Author(s):  
S Horn ◽  
N Bashan ◽  
J Gopas
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Fold Increase ◽  
Fc Receptor ◽  
Autologous Serum ◽  
Murine Macrophages

In this study, the role of Igs in the recognition and removal of oxidatively damaged human red blood cells (RBCs) was investigated. Phagocytosis of normal RBCs exposed to the oxidative hemolytic agent phenylhydrazine (Phz) and of glucose-6-phosphate dehydrogenase (G6PD)- deficient RBCs by murine macrophages was examined. A 40-fold increase in phagocytosis of RBCs treated with 3 mmol/L Phz was obtained both in the absence and presence of autologous serum, indicating that binding of autologous antibodies to the oxidized cells is not essential for phagocytosis. Yet, a basal number of IgG molecules was found to be present on the RBCs, as determined both by binding of 125I protein A and fluorescein isothiocyanate-antihuman Ig to the cells. Macrophage Fc receptors were found to be involved in the recognition of the RBCs, because phagocytosis was partially inhibited by incubating macrophages with bovine serum albumin (BSA) anti-BSA complexes, aIg (aggregated Igs), and anti-Fc receptor II monoclonal antibodies. Galactose/mannose inhibited phagocytosis of oxidized RBCs additively to aIg. Because phagocytosis was decreased when Phz-RBCs were incubated with F(ab')2 fragments of antihuman antibodies, it is suggested that the basal amount of Igs bound to the cells plays a role in the recognition of Phz- RBCs. G6PD-deficient RBCs were recognized and phagocytosed by murine macrophages without preexposure to oxidants in vitro (mean of 19 RBCs/100 macrophages). This phagocytosis was not affected by the addition of serum and was inhibited by incubating macrophages with galactose/mannose and the various Fc receptor blockers. A positive correlation between hemoglobin content and the number of cell-bound Igs to each patient erythrocytes was found. These results support the involvement of both an Fc and a lectin-like macrophage receptor in the recognition and phagocytosis of Phz-oxidized and G6PD-deficient RBCs and suggest opsonization as a possible physiologic process for the removal of severe damaged RBCs.

Src and phosphatidylinositol 3–kinase mediate soluble E-selectin–induced angiogenesis

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3960-3968 ◽  
Author(s):  
Pawan Kumar ◽  
Mohammad A. Amin ◽  
Lisa A. Harlow ◽  
Peter J. Polverini ◽  
Alisa E. Koch
Keyword(s):  
Fold Increase ◽  
Tube Formation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Microvascular Endothelial Cell ◽  
Vitro Assay ◽  
Pi3k Inhibitor Ly294002 ◽  
Soluble E Selectin

Abstract Angiogenesis plays an important role in a variety of pathophysiologic processes, including tumor growth and rheumatoid arthritis. We have previously shown that soluble E-selectin (sE-selectin) is an important angiogenic mediator. However, the mechanism by which sE-selectin mediates angiogenesis is still unknown. In this study, we show that sE-selectin is a potent mediator of human dermal microvascular endothelial cell (HMVEC) chemotaxis, which is predominantly mediated through the Src and the phosphatidylinositiol 3–kinase (PI3K) pathways. Further, sE-selectin induced a 2.2-fold increase in HMVEC tube formation in the Matrigel in vitro assay. HMVECs pretreated with the Src inhibitor (PP2) and the PI3K inhibitor (LY294002) or transfected with Src antisense oligonucleotides or Akt dominant-negative mutants significantly inhibited sE-selectin–mediated HMVEC tube formation. In contrast, HMVECs transfected with an extracellular signal-related kinase 1/2 (ERK1/2) mutant or pretreated with the mitogen-activated protein kinase (MAPK) inhibitor PD98059 failed to show sE-selectin–mediated HMVEC tube formation. Similarly, in the Matrigel-plug in vivo assay, sE-selectin induced a 2.2-fold increase in blood vessel formation, which was significantly inhibited by PP2 and LY294002 but not by PD98059. sE-selectin induced a marked increase in Src, ERK1/2, and PI3K phosphorylation. PI3K and ERK1/2 phosphorylation was significantly inhibited by PP2, thereby suggesting that both of these pathways may be activated via Src kinase. Even though the ERK1/2 pathway was activated by sE-selectin in HMVECs, it seems not to be essential for sE-selectin–mediated angiogenesis. Taken together, our data clearly show that sE-selectin–induced angiogenesis is predominantly mediated through the Src-PI3K pathway.

scholarly journals Basic β-1,3-Glucanase from Drosera binata Exhibits Antifungal Potential in Transgenic Tobacco Plants

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1747
Author(s):  
Miroslav Rajninec ◽  
Monika Fratrikova ◽  
Eva Boszoradova ◽  
Martin Jopcik ◽  
Miroslav Bauer ◽  
...  
Keyword(s):  
Transgenic Tobacco ◽  
Crude Protein ◽  
Trichoderma Viride ◽  
Alternaria Solani ◽  
Fold Increase ◽  
Transcript Abundance ◽  
Carnivorous Plant ◽  
Vitro Assay ◽  
Antifungal Efficiency

The basic β-1,3-glucanase of the carnivorous plant Drosera binata was tested as a purified protein, as well as under the control of a double CaMV35S promoter in transgenic tobacco for its capability to inhibit the growth of Trichoderma viride, Rhizoctonia solani, Alternaria solani, and Fusarium poae in an in-vitro assay. The purified protein inhibited tested phytopathogens but not the saprophytic fungus T. viride. Out of the analysed transgenic plants, lines 13, 16, 19, and 22 exhibited high DbGluc1 transcript abundance normalised to the actin transcript. Because of DbGluc1 transgene expression, lines 13 and 16 showed a 1.7-fold increase and lines 19 and 22 showed more than a 2-fold increase in total β-1,3-glucanase activity compared to the non-transgenic control. In accordance with the purified β-1,3-glucanase in-vitro antifungal assay, crude protein extracts of lines 19 and 22 significantly inhibited the growth of phytopathogens (14–34%). Further analyses revealed that the complementary action of transgenic β-1,3-glucanase and 20% higher activity of endogenous chitinase(s) in these lines were crucial for maximising the antifungal efficiency of crude protein extracts.

scholarly journals Increased visualization of microtubules by an improved fixation procedure.

1977 ◽  
Vol 25 (3) ◽  
pp. 175-187 ◽  
Author(s):  
R B Luftig ◽  
P N McMillan ◽  
J A Weatherbee ◽  
R R Weihing
Keyword(s):  
Phosphate Buffer ◽  
Fold Increase ◽  
Contour Length ◽  
Tetraacetic Acid ◽  
Chick Brain ◽  
Fixation Procedure ◽  
Thin Sections ◽  
Ethanesulfonic Acid ◽  
Brain Microtubules

We have found that when a buffer utilized for in vitro polymerization of microtubules, i.e., 1 mM guanosine triphosphate, 1 mM MgSO4, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.9 polymerization mix, was used in the glutaraldehyde prefixation regimen instead of classical fixative buffers, i.e., isotonic cacodylate or phosphate buffer, the following features were observed in thin-sections of the cytoplasm of interphase HeLa cells: (a) a greater than 2-fold increase in total microtubule contour length, (b) a 2-fold increase in a number of microtubules greater than or equal to 1 mu long, (c) an enhanced association of microtubules with cytoplasmic organelles, and (d) an increased clustering of 100 A filaments located in a perinuclear region of the cell. Furthermore, we found that after we incubated purified chick brain microtubules on a Sephadex G-25 column pre-equilibrated with polymerization mix, cacodylate or phosphate buffer at 37 degrees C, and then eluted the microtubules at 37 degrees C, the exposure to cacodylate or phosphate buffer caused extensive depolymerization, but exposure to polymerization mix buffer allowed reisolation of highly polymerized microtubules. Our results imply that prefixation with cacodylate or phosphate buffered glutaraldenyde destabilizes microtubules leading to the decreased visualization of microtubules.

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