scholarly journals Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples

Cytometry ◽  
2003 ◽  
Vol 55B (1) ◽  
pp. 14-21 ◽  
Author(s):  
Marjolein A. van der Pol ◽  
Henk J. Broxterman ◽  
Guus Westra ◽  
Gert J. Ossenkoppele ◽  
Gerrit J. Schuurhuis
Keyword(s):  
Flow Cytometry ◽  
Simultaneous Detection ◽  
Clinical Samples ◽  
Multiparameter Flow Cytometry ◽  
Flow Cytometry Assay ◽  
P Glycoprotein ◽  
Early Apoptosis

10 A multiparameter flow cytometry assay to monitor natural killer cell proliferation and activation in immuno-oncology clinical trials

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A10-A10
Author(s):  
Jennifer Tsau ◽  
Brittney Atzmiller ◽  
David Quinn ◽  
Tanya Mulvey ◽  
Sema Kurtulus ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cell ◽  
Natural Killer ◽  
Nk Cell ◽  
Multiparameter Flow Cytometry ◽  
Parent Population ◽  
Flow Cytometry Assay ◽  
Cd Markers

BackgroundNatural Killer (NK) cells have garnered increasing interest as potential cellular therapies or as targets of biotherapeutic agents due to their ability to kill tumor cells in a non-antigen dependent manner. Hence, measurement of NK cell proliferation and/or activation following treatment can serve as a useful biomarker for assessing the efficacy of immunomodulatory therapies.MethodsWe developed a novel 13-parameter flow cytometry panel incorporating cell differentiation (CD) markers important for identification of NK cell subsets (CD56, CD16), their proliferation (Ki-67), activation (CD25, CD335, NKG2D) and inhibition (CD159a) status. Additionally, CD markers that identify other cellular subsets known to be amenable to cytokine modulation (e.g., CD3 and CD14) were included for concurrent monitoring of T cell proliferation and monocyte activation. Method validation focused on analytical sensitivity, specificity and precision as key criteria of assay performance using peripheral blood mononuclear cells (PBMCs) stimulated with NK cell-activating cytokines and resting PBMCs from healthy donors.ResultsThe assay design allowed for robust quantitation of NK cell, T cell and monocyte functionalities. Lower limit of quantification (LLOQ) of target biomarker population was determined to be 1.0% of the parent population, based upon an analysis of 110 key target populations that displayed a co-efficient of variation (CV) of ≤25% and their frequencies ranged from 0.1% to 97.8% of the parent population. Additionally, ≤25% CV was observed in precision assessments, confirming the repeatability and reproducibility of the assay. Clinical trial utility of the assay was verified on cryopreserved PBMCs from patients with a variety of solid tumor malignancies. In these patients, the assay could clearly identify proliferating and activated NK cells, as well as proliferating T cells and activated monocytes, thus demonstrating its suitability for clinical trial applications.ConclusionsWe developed and validated a novel multiparameter flow cytometry assay that allows for simultaneous measurement of proliferation, activation and inhibitory status of key immune cell subsets. Thus, this assay can help shed light on the mode of efficacy of novel therapeutic agents that modulate the immune system, aimed at treatment of cancer and autoimmune diseases.

scholarly journals Next-generation multiparameter flow cytometry assay improves the assessment of oxidative stress in probiotics

2020 ◽  
Vol 91 ◽  
pp. 103501
Author(s):  
Vincenzo Fallico ◽  
Mary Rea ◽  
Catherine Stanton ◽  
Niclas Ilestam ◽  
Julie McKinney
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Multiparameter Flow Cytometry ◽  
Next Generation ◽  
Flow Cytometry Assay

scholarly journals Comparative evaluation of S9788, verapamil, and cyclosporine A in K562 human leukemia cell lines and in P-glycoprotein-expressing samples from patients with hematologic malignancies

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 262-269 ◽  
Author(s):  
JL Merlin ◽  
A Guerci ◽  
S Marchal ◽  
N Missoum ◽  
C Ramacci ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cyclosporine A ◽  
K562 Cell ◽  
Simultaneous Detection ◽  
Leukemia Cell ◽  
Hematologic Malignancies ◽  
Clinical Samples ◽  
Human Leukemia ◽  
P Glycoprotein ◽  
Complete Reversal

Abstract The activity of S9788, recently synthetized as a modulator of multidrug resistance (MDR), was compared with verapamil and cyclosporine A in normal sensitive and MDR K562 cell lines, then in samples from 33 patients with hematological malignancies, using flow cytometry with simultaneous detection of P-glycoprotein and determination of intracellular daunorubicin fluorescence. This technique was compared and correlated with a tritiated daunorubicin accumulation method. In K562 cell lines, S9788 exhibited a significantly higher reversing activity than verapamil and cyclosporine A, and allowed a complete restoration of the accumulation of daunorubicin when used at 5 mumol/L. In the clinical samples, the three compounds were evaluated at equimolar concentration (5 mumol/L) using concomitant exposure to daunorubicin and to the reversing agent. In P-glycoprotein-negative samples, no significant effect on intracellular daunorubicin fluorescence of any of the reversing agents was noted. In the 15 P- glycoprotein-positive samples, a significant increase in daunorubicin fluorescence, by at least one reversing agent, was seen in 10 cases, among which S9788 reversing activity was higher than that of the two other agents in seven cases. Complete reversal was only achieved in one case with S9788.

scholarly journals Multiparameter Flow Cytometry Assay for Analysis of Nitrosative Stress Status in Human Spermatozoa

2020 ◽  
Vol 97 (12) ◽  
pp. 1238-1247 ◽  
Author(s):  
Pamela Uribe ◽  
Juan Meriño ◽  
Emilio Manquemilla ◽  
Camila Villagrán ◽  
Etelinda Vega ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nitrosative Stress ◽  
Human Spermatozoa ◽  
Multiparameter Flow Cytometry ◽  
Flow Cytometry Assay

scholarly journals Simultaneous detection of apoptosis and proliferation in colorectal carcinoma by multiparameter flow cytometry allows separation of high and low-turnover tumors with distinct clinical outcome

Cancer ◽  
2003 ◽  
Vol 97 (10) ◽  
pp. 2404-2411 ◽  
Author(s):  
J. Dennis Rupa ◽  
Adriaan P. de Bruïne ◽  
Andrea J. Gerbers ◽  
Mathie P. G. Leers ◽  
Marius Nap ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Colorectal Carcinoma ◽  
Clinical Outcome ◽  
Simultaneous Detection ◽  
Multiparameter Flow Cytometry

scholarly journals Comparative evaluation of S9788, verapamil, and cyclosporine A in K562 human leukemia cell lines and in P-glycoprotein-expressing samples from patients with hematologic malignancies

Blood ◽  
1994 ◽  
Vol 84 (1) ◽  
pp. 262-269
Author(s):  
JL Merlin ◽  
A Guerci ◽  
S Marchal ◽  
N Missoum ◽  
C Ramacci ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cyclosporine A ◽  
K562 Cell ◽  
Simultaneous Detection ◽  
Leukemia Cell ◽  
Hematologic Malignancies ◽  
Clinical Samples ◽  
Human Leukemia ◽  
P Glycoprotein ◽  
Complete Reversal

The activity of S9788, recently synthetized as a modulator of multidrug resistance (MDR), was compared with verapamil and cyclosporine A in normal sensitive and MDR K562 cell lines, then in samples from 33 patients with hematological malignancies, using flow cytometry with simultaneous detection of P-glycoprotein and determination of intracellular daunorubicin fluorescence. This technique was compared and correlated with a tritiated daunorubicin accumulation method. In K562 cell lines, S9788 exhibited a significantly higher reversing activity than verapamil and cyclosporine A, and allowed a complete restoration of the accumulation of daunorubicin when used at 5 mumol/L. In the clinical samples, the three compounds were evaluated at equimolar concentration (5 mumol/L) using concomitant exposure to daunorubicin and to the reversing agent. In P-glycoprotein-negative samples, no significant effect on intracellular daunorubicin fluorescence of any of the reversing agents was noted. In the 15 P- glycoprotein-positive samples, a significant increase in daunorubicin fluorescence, by at least one reversing agent, was seen in 10 cases, among which S9788 reversing activity was higher than that of the two other agents in seven cases. Complete reversal was only achieved in one case with S9788.

Whole Spleen Flow Cytometry Assay

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (15) ◽  
Author(s):  
Cathy Yam ◽  
Adeline Hajjar
Keyword(s):  
Flow Cytometry ◽  
Flow Cytometry Assay

scholarly journals Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 317-327 ◽  
Author(s):  
Martyna Łupicka ◽  
Gabriel Bodek ◽  
Nahum Shpigel ◽  
Ehud Elnekave ◽  
Anna J Korzekwa
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Mrna Expression ◽  
Uterine Horn ◽  
Uterine Tissue ◽  
Pluripotent Cells ◽  
Flow Cytometry Assay ◽  
Myometrial Cells ◽  
Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

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