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.

scholarly journals Role of Capsule and Interleukin-6 in Long-Term Immune Control of Cryptococcus neoformans Infection by Specifically Activated Human Peripheral Blood Mononuclear Cells

2006 ◽  
Vol 74 (9) ◽  
pp. 5302-5310 ◽  
Author(s):  
Asna A. Siddiqui ◽  
Robin J. Shattock ◽  
Thomas S. Harrison
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Interleukin 6 ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Immune Control ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

ABSTRACT Cryptococcus neoformans is a frequent cause of meningoencephalitis in immunosuppressed individuals. To better understand the mechanisms of a protective immune response to C. neoformans, a long-term in vitro model of human immune control of cryptococcal infection was developed. Peripheral blood mononuclear cells (PBMC) prestimulated with heat-killed C. neoformans significantly restricted the growth of C. neoformans after a subsequent live infection compared to that with unstimulated PBMC. Live infection with encapsulated C. neoformans was controlled for as long as 10 days, while infection with acapsular organisms could sometimes be eradicated. During immune control, fungal cells were both intracellular and extracellular within aggregates of mononuclear phagocytes and lymphocytes. Optimal immune control depended on the presence of both CD4+ and CD8+ T cells. Immune control of cryptococcal growth was more effective following prestimulation with acapsular compared with encapsulated organisms. Prestimulation with acapsular organisms was associated with a significant and prolonged increase in interleukin-6 (IL-6) production compared with prestimulation with encapsulated C. neoformans. Addition of IL-6 and depletion of CD25+ T cells prior to prestimulation and infection with encapsulated organisms resulted in reductions in cryptococcal growth that reached borderline statistical significance. Depletion of CD25+ T cells significantly reduced cryptococcal growth in wells with unstimulated PBMC. The results demonstrate an association between high levels of IL-6 and resistance to infection and, through suppression of IL-6 release, an additional mechanism whereby the cryptococcal capsule subverts a protective immune response. Further work is required to clarify the mechanism of action of IL-6 in this setting and any interaction with regulatory T cells.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Emanuela Rasini ◽  
Maria Giulia Albizzati ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Brefeldin A ◽  
Intracellular Cytokine ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

Immunomodulating Effects of "Tien-Hsien Liquid" on Peripheral Blood Mononuclear Cells and T-Lymphocytes from Patients with Recurrent Aphthous Ulcerations

2004 ◽  
Vol 32 (02) ◽  
pp. 221-234 ◽  
Author(s):  
Andy Sun ◽  
Jean-San Chia ◽  
Won-Bo Wang ◽  
Chun-Pin Chiang
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Staphylococcal Enterotoxin B ◽  
Chinese Medicinal Herbs ◽  
Peripheral Blood Mononuclear ◽  
Mucosal Disease ◽  
Blood Mononuclear Cells ◽  
Proliferation Response

Recurrent aphthous ulcerations (RAU) represent a common oral mucosal disease with altered humoral and cellular immunities. In our institution, an immunomodulating agent, levamisole, is used to treat RAU with satisfactory therapeutic effect. Tien-Hsien liquid (THL) is an extract of Chinese medicinal herbs with immunomodulating effects. To test whether THL has immunomodulating effects on antigen-stimulated proliferation response (PR) of peripheral blood mononuclear cells (PBMC) and T-cells isolated from RAU patients and to test whether THL is a potential drug for treating RAU, PBMC or T-cells isolated from RAU patients were incubated with lipopolysaccharides (LPS) from Escherica coli, phytohemagglutinin (PHA), staphylococcal enterotoxin B (SEB), glutaraldehyde-inactivated tetanus toxoid (TT), glucosyltransferase D (GtfD), or antigens of Streptococcus mutans in the presence or absence of THL. We found that THL significantly increased the LPS-stimulated PR of PBMC from active RAU patients, the GtfD-stimulated PR of PBMC and of T-cells from inactive RAU patients, and the S. mutans-stimulated PR of PBMC from inactive RAU patients. However, THL could also significantly reduce the SEB-stimulated PR of PBMC and of T-cells from active RAU patients and S. mutans-stimulated PR of T-cells from active RAU patients. These results suggest that THL can modulate the antigen-stimulated PR of PBMC and T-cells from RAU patients. Therefore, it may be a potential immunoceutical agent for treatment of RAU.

scholarly journals Role of T cells in intrauterine administration of activated peripheral blood mononuclear cells in recurrent implantation failure.

2021 ◽  
Author(s):  
Guillaume Ricaud ◽  
Cathy Vaillancourt ◽  
Veronique Blais ◽  
Marjorie Disdier ◽  
Fabien Joao ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Implantation Failure ◽  
Recurrent Implantation Failure ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Intrauterine administration of autologous peripheral blood mononuclear cells (PBMC) has been recently proposed as new immunotherapy for patients with unexplained recurrent implantation failure (RIF). In these patients, administration of activated PBMC 24-h or 72-h before embryo transfer resulted in a 3-fold increase in biochemical pregnancy rate. In this study we evaluated the role of T cells to promotes human endometrial receptivity. On the day of ovulation, PBMC were isolated from and activated with T cells mitogen, the phytohemagglutinin (PHA) and hCG for 48-h in a conditioned culture medium. Distributions of CD4+ T cells were characterized in 157 patients by flow cytometry before and after PHA/hCG activation. Cytokine production was analyzed by cytometric beads array. We observed in RIF patients a significant decrease in Th2 and natural Treg cells before activation with PHA/hCG and an increase of Th17 cells after activation compared to intrauterine sperm insemination (IUI) and in vitro fertilization (IVF) groups. Furthermore, the hCG/PHA treatment increases anti-inflammatory T cells (Th2 and Treg cells) compared to non-treated T cells. Principal component analysis (PCA) performed on CD4 T cell subtypes revealed a different cellular profile in the RIF compared to the IUI and IVF groups. This inflammatory state change could explain how endometrium immunomodulation by hCG-activated PBMC helps patients with unexplained RIF to reach implantation.

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