Optimization and application of a flow cytometric PU.1 assay for murine immune cells

Immune activation post-myocardial infarction is an orchestrated sequence of cellular responses to effect tissue repair and healing. However, excessive and dysregulated inflammation can result in left ventricular remodeling and pathological alterations in the structural and mechanical attributes of the heart. Identification of key pathways and critical cellular mediators of inflammation is thus essential to design immunomodulatory therapies for myocardial infarction and ischemic heart failure. Despite this, the experimental approaches to isolate mononuclear cells from the heart are diverse, and detailed protocols to enable maximum yield of live cells in the shortest time possible are not readily available. Here, we describe optimized protocols for the isolation, fixation, and flow cytometric characterization of cardiac CD45+ leukocytes. These protocols circumvent time-consuming coronary perfusion and density-mediated cell-separation steps, resulting in high cellular yields from cardiac digests devoid of contaminating intravascular cells. Moreover, in contrast to methanol and acetone, we show that cell fixation using 1% paraformaldehyde is most optimal as it does not affect antibody binding or cellular morphology, thereby providing a considerable advantage to study activation/infiltration-associated changes in cellular granularity and size. These are highly versatile methods that can easily be streamlined for studies requiring simultaneous isolation of immune cells from different tissues or deployment in studies containing a large cohort of samples with time-sensitive constraints. NEW & NOTEWORTHY In this article, we describe optimized protocols for the isolation, fixation, and flow cytometric analysis of immune cells from the ischemic/nonischemic hearts. These protocols are optimized to process several samples/tissues, simultaneously enabling maximal yield of immune cells in the shortest time possible. We show that the low-speed centrifugation can be used as an effective alternative to lengthy coronary perfusion to remove intravascular cells, and sieving through 40-μm filter can replace density-mediated mononuclear cell separation which usually results in 50–70% cell loss in the sedimented pellets. We also show that cell fixation using 1% paraformaldehyde is better than the organic solvents such as methanol and acetone for flow cytometric analysis.

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Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice

The Journal of Immunology ◽

10.4049/jimmunol.2000656 ◽

2021 ◽

pp. ji2000656

Author(s):

Peter M. Carlson ◽

Manasi Mohan ◽

Ravi B. Patel ◽

Jen Birstler ◽

Lauren Nettenstrom ◽

...

Keyword(s):

Immune Cells ◽

Flow Cytometric Analysis ◽

Tumor Bearing ◽

Flow Cytometric

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Histological enzyme and flow cytometric analysis of channel catfish intestinal tract immune cells

Developmental & Comparative Immunology ◽

10.1016/s0145-305x(01)00044-1 ◽

2002 ◽

Vol 26 (1) ◽

pp. 53-62 ◽

Cited By ~ 18

Author(s):

Pamela Hébert ◽

A.Jerald Ainsworth ◽

Bobbie Boyd

Keyword(s):

Immune Cells ◽

Channel Catfish ◽

Intestinal Tract ◽

Flow Cytometric Analysis ◽

Flow Cytometric

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Salt-sensitive increase in macrophages in the kidneys of Dahl SS rats

AJP Renal Physiology ◽

10.1152/ajprenal.00096.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. F361-F374 ◽

Cited By ~ 14

Author(s):

Daniel J. Fehrenbach ◽

Justine M. Abais-Battad ◽

John Henry Dasinger ◽

Hayley Lund ◽

David L. Mattson

Keyword(s):

Immune Cells ◽

Renal Damage ◽

Flow Cytometric Analysis ◽

Toll Like Receptor ◽

Sprague Dawley ◽

Dietary Salt ◽

Renal Hypertrophy ◽

Flow Cytometric ◽

Splenic Macrophages ◽

Salt Sensitive Hypertension

Studies of Dahl salt-sensitive (SS) rats have shown that renal CD3+ T cells and ED-1+ macrophages are involved in the development of salt-sensitive hypertension and renal damage. The present study demonstrated that the increase in renal immune cells, which accompanies renal hypertrophy and albuminuria in high-salt diet-fed Dahl SS rats, is absent in Sprague-Dawley and SSBN13 rats that are protected from the SS disease phenotype. Flow cytometric analysis demonstrated that >70% of the immune cells in the SS kidney are M1 macrophages. PCR profiling of renal myeloid cells showed a salt-induced upregulation in 9 of 84 genes related to Toll-like receptor signaling, with notable upregulation of the Toll-like receptor 4/CD14/MD2 complex. Because of the prominent increase in macrophages in the SS kidney, we used liposome-encapsulated clodronate (Clod) to deplete macrophages and assess their contribution to salt-sensitive hypertension and renal damage. Dahl SS animals were administered either Clod-containing liposomes (Clod-Lipo), Clod, or PBS-containing liposomes as a vehicle control. Clod-Lipo treatment depleted circulating and splenic macrophages by ∼50%; however, contrary to our hypothesis, Clod-Lipo-treated animals developed an exacerbated salt-sensitive response with respect to blood pressure and albuminuria, which was accompanied by increased renal T and B cells. Interestingly, those treated with Clod also demonstrated an exacerbated phenotype, but it was less severe than Clod-Lipo-treated animals and independent of changes to the number of renal immune cells. Here, we have shown that renal macrophages in Dahl SS animals sustain a M1 proinflammatory phenotype in response to increased dietary salt and highlighted potential adverse effects of Clod-Lipo macrophage depletion.

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Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Journal of Visualized Experiments ◽

10.3791/53658 ◽

2016 ◽

Cited By ~ 15

Author(s):

Claudia Pösel ◽

Karoline Möller ◽

Johannes Boltze ◽

Daniel-Christoph Wagner ◽

Gesa Weise

Keyword(s):

Mouse Brain ◽

Immune Cells ◽

Flow Cytometric Analysis ◽

Flow Cytometric

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Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106786118 ◽

2021 ◽

Vol 118 (28) ◽

pp. e2106786118

Author(s):

Darui Xu ◽

Stephen Lyon ◽

Chun Hui Bu ◽

Sara Hildebrand ◽

Jin Huk Choi ◽

...

Keyword(s):

Machine Learning ◽

Immune Cells ◽

Linkage Data ◽

Mapping Data ◽

Genetic Studies ◽

Flow Cytometric ◽

Linkage Information ◽

Meiotic Mapping ◽

Immune Phenotypes ◽

Learning Software

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation–phenotype association. At this time, CE has evaluated putative mutation–phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

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A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

PLoS ONE ◽

10.1371/journal.pone.0150606 ◽

2016 ◽

Vol 11 (3) ◽

pp. e0150606 ◽

Cited By ~ 115

Author(s):

Yen-Rei A. Yu ◽

Emily G. O’Koren ◽

Danielle F. Hotten ◽

Matthew J. Kan ◽

David Kopin ◽

...

Keyword(s):

Immune Cells ◽

Flow Cytometric Analysis ◽

Lymphoid Tissues ◽

Flow Cytometric

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Simmental × Holstein crossbred: comparison of immunological traits with parental breeds during peripartum and early lactation period

Journal of Dairy Research ◽

10.1017/s0022029920000928 ◽

2020 ◽

pp. 1-5

Author(s):

Maria Carmela Scatà ◽

Francesco Grandoni ◽

Vittoria Lucia Barile ◽

Gennaro Catillo ◽

Giovanna De Matteis

Keyword(s):

T Lymphocytes ◽

B Lymphocytes ◽

Immune Cells ◽

Lower Percentage ◽

Lactation Period ◽

Early Lactation ◽

Crossbred Cows ◽

Adaptive Immune ◽

Flow Cytometric ◽

Adaptive Immune Cells

Abstract The experiment described in this research communication aimed to compare the immunological traits of Simmental (sire) × Holstein (dam) crossbred cows with the two parental breeds in the peripartum and early lactation period and to estimate the effects of heterosis for these traits. Flow cytometric evaluation of leukocyte subpopulations was assessed in 16 Crossbred (CR), 8 Holstein (HO) and 8 Simmental (SI) cows. Estimated average values of innate and adaptive immune cells showed statistically significant differences between the crossbred cows and parental breeds. Interestingly, the most relevant differences between the three groups related to adaptive immune cells. In particular, the CR cows showed a lower percentage of CD3+ T lymphocytes compared with the SI group (P < 0.0001) and the highest proportions of CD21+ B lymphocytes among the three groups (P < 0.0001). Furthermore, we found the highest positive value of heterosis for the CD21+ B lymphocytes (7.0) and the lowest negative value for CD3+ T lymphocytes (−4.8) in F1 derived population. It seems reasonable to believe that these differences could affect immune function of crossbred cows.

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Effects of 10-day confinement on the immune system and psychological aspects in humans

Journal of Applied Physiology ◽

10.1152/japplphysiol.00043.2004 ◽

2004 ◽

Vol 97 (3) ◽

pp. 920-924 ◽

Cited By ~ 17

Author(s):

Tamiyasu Shimamiya ◽

Nobuyuki Terada ◽

Yoshimitsu Hiejima ◽

Sonoe Wakabayashi ◽

Hirotake Kasai ◽

...

Keyword(s):

Nk Cells ◽

Immune Cells ◽

Psychological Aspects ◽

Flow Cytometric ◽

Face Scale ◽

The Face ◽

Scale Test ◽

Confined Environment ◽

Immune Changes ◽

Physiological Reactions

We investigated the changes in percentages of leukocyte subpopulations, natural killer (NK) cells, CD69-expressing lymphocytes, and psychological aspects in 10 subjects who participated in a 10-day confinement study. Suppression of lymphocyte proliferative reaction and changes in leukocyte distribution are known to occur in space. These responses are similar to those induced by psychological stress. Ground-based confinement studies are suitable for validating the effects of stress arising only due to confinement. Two groups, consisting of five male subjects (ages 20–27 yr, mean 22.8 yr) each, participated in a 10-day confinement study. Blood samples were taken once before, three times during, and once after the confinement and activated with an anti-CD2 agonistic antibody cocktail. The percentages of leukocyte subpopulations, NK (CD45+CD56+) cells, and activated lymphocytes (CD45+CD69+) were measured by flow cytometric assay. The face scale test was used to measure psychological aspects. The percentage of CD69+ lymphocytes decreased during the period of confinement. This was mostly caused by changes in the ratio between NK and non-NK lymphocytes. The face scale showed that the subjects' moods improved toward the postconfinement period. Consistent with the face scale, the percentages of innate immune cells, such as NK cells and granulocytes, increased during the postconfinement period. We concluded that the changes in the distribution of immune cells caused by stress plays an important role in suppression of proliferative reactivity. The observed physiological reactions were specific to the confined environment, and the stress caused by confinement plays a role in the immune changes observed in space.

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