scholarly journals Anti-human Interleukin(IL)-4 Clone 8D4-8 Cross-Reacts With Myosin-9 Associated With Apoptotic Cells and Should Not Be Used for Flow Cytometry Applications Querying IL-4 Expression

2019 ◽  
Vol 7 ◽  
Author(s):  
Robert Z. Harms ◽  
Kiana Borengasser ◽  
Vikas Kumar ◽  
Nora Sarvetnick
Keyword(s):  
Flow Cytometry ◽  
Apoptotic Cells

Monitoring circulating apoptotic cells by in-vivo flow cytometry

2008 ◽  
Author(s):  
Xunbin Wei ◽  
Yuan Tan ◽  
Yun Chen ◽  
Li Zhang ◽  
Yan Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Apoptotic Cells ◽  
In Vivo Flow Cytometry

The Plasticity of Myeloma Plasma Cells as Expressed by Dedifferentiation into an Immature, Resilient, Apoptosis-Resistant Phenotype.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 633-633 ◽  
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Guido Tricot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rabbit Model ◽  
Common Mechanism ◽  
Proliferative Potential ◽  
Apoptotic Cells ◽  
Apoptosis Resistance ◽  
Hematopoietic Stem ◽  
Induced Apoptosis

Abstract Progression of myeloma (MM) is considered to be a multistage and dynamic process of cell differentiation, survival, proliferation and dissemination. We have previously demonstrated the proliferative potential of purified CD45lowCD38high mature MM cells in SCID-hu mice (Yaccoby & Epstein, Blood, 1999), the ability of CD138-selected MM cells to produce myeloma in our novel SCID-rabbit model (Yata et al., ASH, 2003), and the interdependence of MM bone disease and tumor growth whereby MM cells induce osteoclast activity and are dependent on osteoclasts in vivo (Yaccoby et al., BJH, 2002) and ex vivo (Yaccoby et al., Cancer Res., 2004). The aim of this study was to determine the osteoclast-induced phenotypic changes associated with survival of MM cells in long term co-culture. CD138-selected (>95% purity) MM cells from 16 patients were co-cultured with human osteoclasts for up to 20 weeks. The pre-cultured baseline cells were typically CD45low/inermediateCD38high, CD19−CD34−. At the end of long term co-culture (>6 weeks) MM cells had BrdU labeling index (LI) of 2.5±2.0 and their viability was 97%±1%. The phenotype of co-cultured MM cells consistently shifted to a less mature phenotype, with CD45 expression increasing from CD45low to CD45intermmediata/high and reduced expression of CD38 from CD38high to subpopulations with CD38intermediate levels, as determined by flow cytometry and confirmed by qRT-PCR. Further flow analysis revealed that co cultured MM cells also expressed low levels of CD19 and CD34, and identified a small subpopulation of CD138lowCD45high MM cells. Morphologically, the co-cultured MM cells uniformly gained plasmablastic characteristics when compared to pre-cultured cells. Previous reports suggested that IL-6 was important for maintaining subpopulation of CD45-expressing MM cells. However, blocking IL-6 activity in co-cultures with anti-IL6 and anti-IL6R neutralizing antibodies (5 μg/ml, each) did not affect the immature phenotype of MM cells. Intriguingly, long term co-culture of normal CD34+ hematopoietic stem cells (HSCs) with osteoclasts results in loss of CD34 expression, suggesting a common mechanism for osteoclast-induced MM PC and HSC plasticity. To investigate if the observed phonotypic changes are associated with apoptosis resistance, we determined the effects of 3 days exposure to the pro-apoptotic agent dexamethasone (DEX, 10−7 M) on MM cells cultured alone or in co-cultures (n=5), at initiation (baseline) and after 6 weeks of co-cultures. The percent apoptotic cells was determined by trypan blue exclusion and annexin V flow cytometry. When baseline MM cells were cultured alone, DEX significantly increased the percent of apoptotic cells over that spontaneous rate (p<0.01). In contrast, when MM cells recovered from co-cultures after 6 weeks they survived and were resistant to DEX-induced apoptosis (16%±11% and 24%±21% apoptotic cells in the absence and presence of DEX, respectively). As reported, osteoclasts supported survival of MM cells at baseline and after 6 weeks of co-culture (p<0.01), and protected MM PCs from DEX-induced apoptosis. Our data demonstrate the phenotypic plasticity of primary myeloma cells, whereby mature MM cells are reprogrammed and acquire autonomous survival properties after co-culture with osteoclasts. We hypothesize that in vivo these cells are dormant, resistant to spontaneous and drug-induced apoptosis, and could be responsible for relapse.

scholarly journals Critical Evaluation of Techniques to Detect and Measure Cell Death – Study in a Model of UV Radiation of the Leukaemic Cell Line HL60

1999 ◽  
Vol 19 (3-4) ◽  
pp. 139-151 ◽  
Author(s):  
Marina Leite ◽  
Margarida Quinta‐Costa ◽  
Pedro Simas Leite ◽  
José Eduardo Guimarães
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Fluorescence Microscopy ◽  
Trypan Blue ◽  
Critical Evaluation ◽  
Annexin V ◽  
Tunel Assay ◽  
Apoptotic Cells ◽  
Dna Ladder ◽  
Apoptosis And Necrosis

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V‐FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.

scholarly journals The study of apoptotic cells by flow cytometry

Leukemia ◽  
1998 ◽  
Vol 12 (7) ◽  
pp. 1013-1025 ◽  
Author(s):  
MG Ormerod
Keyword(s):  
Flow Cytometry ◽  
Apoptotic Cells

Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry

2016 ◽  
Vol 2016 (11) ◽  
pp. pdb.prot087312 ◽  
Author(s):  
Lisa C. Crowley ◽  
Nigel J. Waterhouse
Keyword(s):  
Flow Cytometry ◽  
Caspase 3 ◽  
Apoptotic Cells

scholarly journals Anti-Tyro3 IgG associates with disease activity and reduces efferocytosis of macrophages in new-onset systemic lupus erythematosus

2020 ◽  
Author(s):  
Zhuochao Zhou ◽  
Aining Xu ◽  
Jialin Teng ◽  
Fan Wang ◽  
Yun Tan ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Flow Cytometry ◽  
Disease Activity ◽  
Lupus Erythematosus ◽  
Serum Levels ◽  
Enzyme Linked Immunosorbent Assay ◽  
Apoptotic Cells ◽  
Systemic Lupus ◽  
Oral Ulcers ◽  
New Onset

Abstract Backgroud: To investigate the role of Tyro3 receptor in macrophages’ efferocytosis of apoptotic cells in systemic lupus erythematosus (SLE), we aimed to reveal the clinical relevance and impact of anti-Tyro3 antibody on SLE. Methods : The serum levels of IgG-type autoantibody against Tyro3 receptor were detected in new-onset, treatment-naïve SLE patients (n =70) and healthy controls (HCs) (n =70) using enzyme-linked immunosorbent assay (ELISA). The correlation of the levels of autoantibodies against Tyro3 receptor with clinical and laboratory characteristics were analyzed by Spearman correlation analysis. Receiver operating characteristic (ROC) curve was used to assess the sensitivity and specificity of anti-Tyro3 IgG for the diagnosis of SLE. The effects of purified Tyro3 autoantibody from SLE patients on the efferocytosis of human monocyte-derived macrophages were measured by flow cytometry and immunofluorescence. Results : The serum levels of IgG-type autoantibody against Tyro3 receptor were significantly elevated in patients with SLE compared to HCs ( p < 0.0001). The levels of anti-Tyro3 IgG were negatively associated with haemoglobin (Hb) ( r =-0.294, p = 0.014), and positively correlated with the presence of oral ulcers ( r = 0.254, p = 0.034), SLE disease activity index (SLEDAI) score ( r = 0.254, p = 0.034), erythrocyte sedimentation rate (ESR) ( r = 0.430, p = 0.000), C-reactive protein (CRP) ( r = 0.246, p = 0.049) and immunoglobulin G (IgG) ( r = 0.408, p = 0.001). Higher levels of anti-Tyro3 antibody were observed in patients with oral ulcers than paitents without oral ulcers ( p = 0.035). Further flow cytometry demonstrated that purified anti-Tyro3 IgG inhibited the efferocytosis of macrophages ( p = 0.004). Immunofluorescence assay also showed a decreased engulfment of apoptotic cells in the macrophages incubated with purified anti-Tyro3 IgG ( p = 0.044) compared with control IgG. Conclusions: These observations indicated that autoantibody against Tyro3 was associated with disease activity and impaired efferocytosis of macrophages, which might be involved in the pathogenesis of SLE.

The Role of Urokinase Receptor (uPAR) In Efferocytosis: uPAR Engulfs Apoptotic Cells via a Phosphatidylserine Pathway Mediated by Plasma High Molecular Weight Kininogen

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 929-929 ◽  
Author(s):  
Aizhen Yang ◽  
Jihong Dai ◽  
Raymond B. Birge ◽  
Yi Wu
Keyword(s):  
Molecular Weight ◽  
Flow Cytometry ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cells ◽  
High Molecular Weight Kininogen ◽  
Viable Cells

Abstract Abstract 929 Phagocytosis of apoptotic cells by phagocytes, also known as efferocytosis, is essential for maintaining normal tissue homeostasis and regulating immune responses. Defects in rapid clearance of apoptotic cells lead to the release of immunogenic cellular contents, which may cause tissue damage and autoimmune disease. Phagocytic receptors differentiate apoptotic cells from viable cells by recognizing ‘don't eat- or eat-me’ signals on the cell surface. Recently, we and others have reported the role of uPAR in mediating efferocytosis. In this study, we examined the mechanism by which uPAR recognizes and internalizes apoptotic cells. By flow cytometry-based in vivo and in vitro phagocytosis assay, we found that in knockout mice the lack of uPAR expression on macrophages decreased their apoptotic cell engulfing activity by >35%. Conversely, soluble uPAR and polyclonal anti-uPAR antibodies (Ab) suppressed the internalization of apoptotic cells by macrophages. However, there was no defect in uPAR-/- macrophage uptake of viable cells, suggesting that uPAR plays a specific role in phagocytosis of apoptotic cells. We established a HEK 293 cell line expressing human full-length uPAR (293-uPAR). In these cells, uPAR-mediated phagocytosis of apoptotic cells was completely blocked by annexin V in the presence of calcium. The effect of annexin V was not observed in the absence of calcium, indicating that uPAR internalizes apoptotic cells through a phosphatidylserine pathway. We also found that uPAR-mediated uptake of apoptotic cells was completely prevented under serum-free conditions. To identify plasma proteins that may opsonize the uPAR function, we used immunodepletion method to test three known uPAR-binding proteins, vitronectin, uPA and high molecular weight kininogen (HK). Depletion of HK from serum by a polyclonal anti-HK Ab significantly reduced the engulfment of apoptotic cells by either macrophages or 293-uPAR cells in a co-culture system. In contrast, depletion of vitronectin or uPA from serum had little effect. uPAR is a GPI-anchored protein. Upon sucrose gradient ultracentrifugation, the majority of uPAR molecules were co-localized with membrane-bound HK in lipid rafts. The binding capacity of HK to apoptotic cell surface was further analyzed by flow cytometry. Phycoerythrin-labeled HK bound to apoptotic cells in a concentration-dependent manner, saturating at 300 nM. In contrast, HK did not bind to viable cells at concentrations up to 1200 nM. It is known that HK is a key component of the plasma contact system and that apoptotic cells potentiate factor Xa formation. Our new findings of the uPAR-HK-phosphatidylserine axis in efferocytosis suggest that this pathway may modulate the coagulation cascade on the surface of apoptotic cells. This pathway may also play a role in the pathogenesis of autoimmune and thrombotic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Effect of Ceramide on Model Membranes and Apoptotic Cells Determined by X-Ray Scattering, Solid State NMR, and Flow Cytometry

2010 ◽  
Vol 98 (3) ◽  
pp. 465a-466a
Author(s):  
Matthew J. Justice ◽  
Adriana L. Rogozea ◽  
Daniela N. Petrusca ◽  
Irina Petrache ◽  
Stephen R. Wassall ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Solid State ◽  
Solid State Nmr ◽  
Model Membranes ◽  
Apoptotic Cells ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

scholarly journals Polymorphonuclear Leukocyte Apoptosis Is Accelerated by Sulfatides or Sulfatides-TreatedSalmonellaTyphimurium Bacteria

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Zoryana V. Grishina ◽  
Galina M. Viryasova ◽  
Yulia M. Romanova ◽  
Galina F. Sud’ina
Keyword(s):  
Flow Cytometry ◽  
Polymorphonuclear Leukocyte ◽  
Human Neutrophil ◽  
Apoptotic Index ◽  
Neutrophil Apoptosis ◽  
Apoptotic Cells ◽  
Galactosidase Activity ◽  
Leukocyte Apoptosis ◽  
Intracellular Compartments ◽  
Ceramide Content

Neutrophils die by apoptosis following activation and uptake of microbes or enter apoptosis spontaneously at the end of their lifespan if they do not encounter a pathogen. Here we report that sulfatides or sulfatides-treatedSalmonellaTyphimurium bacteria accelerated human neutrophil apoptosis. Neutrophil apoptosis was examined by flow cytometry. Sulfatides caused prominent increase in percentage of apoptotic cells after 2.5 hrs of incubation.SalmonellaTyphimurium bacteria by themselves did not affect the basal level of apoptosis in neutrophil population. When neutrophils were added toS.Typhimurium “opsonized” by sulfatides, apoptotic index significantly increased, whereas the number of phagocyting cells was not influenced. Sulfatides’ proapoptotic effect was strongly dependent on the activity ofβ-galactosidase; inhibition of this enzyme impaired its potency to accelerate apoptosis. These data support the mechanism of neutrophil apoptosis triggering based on sulfatides’ ability to accumulate in intracellular compartments and mediate successive increase in ceramide content resulting fromβ-galactosidase activity.

Sign in / Sign up

Export Citation Format

Share Document