scholarly journals Optimization and Limitations of Use of Cryopreserved Peripheral Blood Mononuclear Cells for Functional and Phenotypic T-Cell Characterization

2009 ◽  
Vol 16 (8) ◽  
pp. 1176-1186 ◽  
Author(s):  
Adriana Weinberg ◽  
Lin-Ye Song ◽  
Cynthia Wilkening ◽  
Anne Sevin ◽  
Bruce Blais ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Pokeweed Mitogen ◽  
Cell Recovery ◽  
Flow Cytometry Assay ◽  
Peripheral Blood Mononuclear ◽  
Affect Cell Viability ◽  
Blood Mononuclear Cells

ABSTRACT The goals of this study were to optimize processing methods of cryopreserved peripheral blood mononuclear cells (PBMC) for immunological assays, identify acceptance parameters for the use of cryopreserved PBMC for functional and phenotypic assays, and to define limitations of the information obtainable with cryopreserved PBMC. Blood samples from 104 volunteers (49 human immunodeficiency virus-infected and 55 uninfected) were used to assess lymphocyte proliferation in response to tetanus, candida, and pokeweed-mitogen stimulation and to enumerate CD4+ and CD8+ T cells and T-cell subpopulations by flow cytometry. We determined that slowly diluting the thawed PBMC significantly improved viable cell recovery, whereas the use of benzonase improved cell recovery only sometimes. Cell storage in liquid nitrogen for up to 15 months did not affect cell viability, recovery, or the results of lymphocyte proliferation assays (LPA) and flow cytometry assays. Storage at −70°C for ≤3 weeks versus storage in liquid nitrogen before shipment on dry ice did not affect cell viability, recovery, or flow cytometric results. Storage at −70°C was associated with slightly higher LPA results with pokeweed-mitogen but not with microbial antigens. Cell viability of 75% was the acceptance parameter for LPA. No other acceptance parameters were found for LPA or flow cytometry assay results for cryopreserved PBMC. Under optimized conditions, LPA and flow cytometry assay results for cryopreserved and fresh PBMC were highly correlated, with the exception of phenotypic assays that used CD45RO or CD62L markers, which seemed labile to freezing and thawing.

scholarly journals Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

2018 ◽  
Vol 51 (2) ◽  
pp. 168-173
Author(s):  
Luzia Aparecida Costa Barreira ◽  
Priscila Santos Scheucher ◽  
Marilia Farignoli Romeiro ◽  
Leonardo La Serra ◽  
Soraya Jabur Badra ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Flow Cytometry Assay ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

scholarly journals PBMC Thawing Protocol v1

2020 ◽  
Author(s):  
Fang Zhang
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Flow Cytometry Assay ◽  
Peripheral Blood Mononuclear ◽  
Cell Staining ◽  
Blood Mononuclear Cells

This protocol details methods for thawing peripheral blood mononuclear cells (PBMC). For a protocol detailing Culture and Stimulation, please view the following: PBMCs Culture and Stimulation. For a protocol detailing Cell Staining for Flow Cytometry Assay, please view the following: Cell Staining for Flow Cytometry Assay.

scholarly journals Flow Cytometry Assay to Monitor Effectiveness of Gene Therapy Correction in Peripheral Blood Mononuclear Cells of Gaucher Disease Type I Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5791-5791
Author(s):  
Cecilia Barese ◽  
Michael Waring ◽  
Richard Pfeifer ◽  
Chris Mason
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Gaucher Disease ◽  
Mononuclear Cells ◽  
Disease Type ◽  
Normal Donor ◽  
Type I ◽  
Flow Cytometry Assay ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract The most common lysosomal storage disorder is Gaucher disease type I (GD-I) which is caused by a genetic defect in the gene encoding the enzyme beta-glucocerebrosidase (GCase). In GD-I, deficient GCase activity results in accumulation of glucocerebrosidases within macrophages of the reticuloendothelial system. We hypothesize that putative precursors for macrophages, monocytes, in peripheral blood of GD-I patients, represent an excellent target population to monitor the effectiveness of novel curative treatments such as lentiviral vector driven gene correction targeting hematopoietic stem and progenitor cells in clinical setting. Here, we present an improved flow cytometry assay that takes advantage of selective uptake of pentafluorobenzoylamino fluorescein di-β-D-glucopyranoside by the CD14+ monocytes resulting in fluorescence when it is hydrolyzed by active GCase, in the presence of formaldehyde (FA), commonly used for intracellular staining and to inactivate pathogens. Using this substrate uptake/FA fixation method our results show a mean of 33.7% ± 3.6% (X±SD) GCase+ cells in GD-1 patients (n=2) compared to 83.7% ± 9.6% (X±SD) in normal controls (n=5). The mean fluorescence intensity (MFI) of the enzymatic activity showed no overlap between untreated GD-1 patients (3480 ± 49) and controls (7187 ± 595.7). Method specificity was demonstrated by complete inhibition of enzyme activity and fluorescence using 400 mM conduritol-B-epoxide, a GCase specific inhibitor. Next, mixtures of normal donor peripheral blood mononuclear cells (PBMC) and GD-1 PBMC were created in vitro to assess the lower limit of detection. As low as 1% normal donor cells could be reliably distinguished within these mixtures. GCase+ monocytes in the mixtures could be identified and quantified based on a higher MFI compared to GD-1 patient cells. Compared to the conventional 4-MU biochemical method, this approach requires only a small blood sample and minimal processing, is rapid and straightforward, and more importantly allows quantification of GCase enzyme activity at individual cells instead of in a bulk cell lysate. These findings suggest this novel flow cytometry method is sensitive and specific to quantify small increases in GCase activity at single cell level, and thus has potential to monitor in vivo the effectiveness of gene therapy for Gaucher disease Type 1 in patients in real time. Additional sample analysis from GD-1 patients and normal donors are in progress to confirm consistency of the method. Disclosures No relevant conflicts of interest to declare.

scholarly journals Viability and Functional Activity of Cryopreserved Mononuclear Cells

2000 ◽  
Vol 7 (4) ◽  
pp. 714-716 ◽  
Author(s):  
Adriana Weinberg ◽  
Li Zhang ◽  
Darby Brown ◽  
Alejo Erice ◽  
Bruce Polsky ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Functional Activity ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Cell Number ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
And Function ◽  
Functional Analyses ◽  
Cd4 Cell

ABSTRACT Factors that influence viability and function of cryopreserved peripheral blood mononuclear cells (PBMC) were identified on 54 samples from 27 AIDS Clinical Trial Units. PBMC viability ranged from 1 to 96% with a median of 70%, was higher in laboratories with experienced staff, and was not significantly associated with CD4 cell number. Function of cryopreserved PBMC, measured by lymphocyte proliferation, was associated with viability. Preparations with viability greater than or equal to 70% had consistent proliferative responses and were suitable for functional analyses.

Traditional Acupuncture Increases the Content of Beta-Endorphin in Immune Cells and Influences Mitogen Induced Proliferation

1991 ◽  
Vol 19 (02) ◽  
pp. 101-104 ◽  
Author(s):  
Mauro Bianchi ◽  
Edda Jotti ◽  
Paola Sacerdote ◽  
Alberto E. Panerai
Keyword(s):  
Immune Responses ◽  
T Lymphocyte ◽  
Peripheral Blood Mononuclear Cells ◽  
Immune Cells ◽  
Lymphocyte Proliferation ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Beta Endorphin ◽  
T Lymphocyte Proliferation ◽  
Blood Mononuclear Cells

We measured beta-endorphin concentrations in peripheral blood mononuclear cells and mitogen-induced T-lymphocyte proliferation in patient who underwent treatment with traditional acupuncture. Traditional acupuncture increased both the concentrations of the opioid in the immune cells and lymphocyte proliferation. Our data are consistent with the hypothesis that traditional acupuncture modulates immune responses in man.

scholarly journals Production of γ-Interferon by Peripheral Blood Mononuclear Cells: An Important Diagnostic Tool for Detection of Subclinical Paratuberculosis

1996 ◽  
Vol 8 (3) ◽  
pp. 345-350 ◽  
Author(s):  
Judith R. Stabel
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Diagnostic Tool ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Pokeweed Mitogen ◽  
Complete Medium ◽  
Similar Response ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Important Diagnostic Tool

Peripheral blood mononuclear cells were isolated from noninfected control cows and from cows with either subclinical or clinical paratuberculosis (Johne s disease). Cells were incubated for 6, 12, 24, and 48 hours in complete medium with the following mitogens: concanavalin A (ConA), phytohemagglutinin-P (PHAP), pokeweed mitogen (PWM), and Escherichia coli lipopolysaccharide. In addition, cells were incubated for the same time periods with a Mycobacterium paratuberculosis sonicate (MpS) and live and heat-killed M. paratuberculosis at 10:1 bacteria: cell ratio. After incubation, cell-free supernatants were analyzed for y-interferon (γ-IFN) production. Cells from subclinical cows produced significantly higher levels of γ-IFN than did cells from clinical animals after stimulation with mitogens ConA, PHAP, and PWM. Levels of γ-IFN produced by noninfected control animals generally followed the pattern of those of subclinical animals. After incubation with MpS, significantly greater quantities of γ-IFN were produced by cells isolated from subclinical animals than by cells from clinical cows and noninfected controls. Stimulation of cells with heat-killed or live M. paratuberculosis evoked a similar response. This study indicates that γ-IFN production by peripheral blood mononuclear cells in response to M. paratuberculosis antigen may be an important diagnostic tool for the detection of paratuberculosis in subclinically affected animals.

scholarly journals Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

2021 ◽  
Author(s):  
Bo Li ◽  
Chunmei Yang ◽  
Gui Ja ◽  
Yansheng Liu ◽  
Na Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Effective Utilization ◽  
Blood Mononuclear Cells ◽  
Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

Alterations in the Distribution and Proliferative Responses of Rhesus Monkey Peripheral Blood and Spleen Cells During Malaria ( Plasmodium knowlesi ) Infection

1980 ◽  
Vol 28 (2) ◽  
pp. 502-507
Author(s):  
Diane W. Taylor ◽  
Suzanne Richmond Crum ◽  
Kenton J. Kramer ◽  
Wasim A. Siddiqui
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Rhesus Monkeys ◽  
Mononuclear Cells ◽  
Plasmodium Knowlesi ◽  
Pokeweed Mitogen ◽  
Spleen Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Rhesus monkeys are used frequently as animal models in malaria research, but few studies have evaluated lymphocyte functions in these animals after experimental infections with the primate malarial parasite Plasmodium knowlesi. In this study, the distribution and mitogen responses of mononuclear cells in the peripheral blood and spleens of 16 P. knowlesi -infected rhesus monkeys were followed. All animals included in the study developed acute infections and were bled out with parasitemias of more than 50%. With progression of the infection, alterations in the peripheral blood mononuclear cells were observed, including decreases in the percentage of T cells (measured by E rosette formation) and the total numbers of E and EAC rosette-forming cells per cubic millimeter. In addition, peripheral blood mononuclear cells displayed reduced responses to mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen. Peripheral blood mononuclear cells from infected animals showed similar reductions in mitogen responses when cultured in media containing 15% autologous pre- or postinfection plasma. The mitogen responses of spleen cells did not appear to be affected, but a significant reduction in the proportion of splenic T cells was observed. These lymphocyte changes in P. knowlesi -infected rhesus monkeys are similar to those reported for mice with acute rodent malaria and for humans with chronic Plasmodium falciparum infections.

scholarly journals Soluble CD16 binds peripheral blood mononuclear cells and inhibits pokeweed-mitogen-induced responses

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3081-3090 ◽  
Author(s):  
C Teillaud ◽  
J Galon ◽  
MT Zilber ◽  
N Mazieres ◽  
R Spagnoli ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Specific Interaction ◽  
Pokeweed Mitogen ◽  
Human Neutrophils ◽  
Induced Responses ◽  
Peripheral Blood Mononuclear ◽  
Extracellular Region ◽  
Blood Mononuclear Cells

Abstract Human neutrophils express two types of low affinity receptors for IgG, Fc gamma RII or CD32 and Fc gamma RIIIB or CD16. Human serum contains soluble CD16 (sCD16), which is produced by proteolysis of neutrophil Fc gamma RIIIB, the cleavage site being located close to the cell surface. In order to assess the functional roles of sCD16, we have produced, in eukaryotic cells, a recombinant sCD16 containing the extracellular region of Fc gamma RIIIB. Purified sCD16, of molecular mass of 48 kD, bound human IgG1 and IgG3 but not IgG2, IgG4, or F(ab')2. It inhibited, in a time and dose-dependent fashion, proliferation and IgM and IgG production of human peripheral blood mononuclear cells (PBMC) stimulated by pokeweed mitogen (PWM) in vitro. FACS analysis showed that biotinylated sCD16 bound specifically to a fraction (35%) of PBMC, which corresponds to monocytes and to subsets of B and T lymphocytes. Moreover, sCD16 did not modify the staining of PBMC by FITC-coupled PWM. Thus, the biologic function(s) of sCD16 on PWM-induced responses are exerted through direct and specific interaction(s) with mononuclear blood cells and not with PWM. In conclusion, neutrophils may play a regulatory role on immune responses via the production of soluble forms of CD16 with cell-binding and antiproliferative capacities.

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