Guidelines for authors and reviewers on antibody use in physiology studies

Brooks, Heddwen L.; Lindsey, Merry L.

doi:10.1152/ajpheart.00512.2017

Cited by 71 publications

(51 citation statements)

References 57 publications

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“…reliable and replicable). The results from these studies informed the generation of a SOP which includes relevant suggestions and observations to assist implementation of best practices in flow cytometry consistent with other recent calls for increased rigor with respect to antibody use (Bordeaux et al, 2010;Bradbury and Pluckthun, 2015;Brooks and Lindsey, 2018).…”

Section: Discussionsupporting

confidence: 66%

“…The spirit of this study is responsive to recent calls for improving scientific rigor and reproducibility discussed in several recent publications (Bordeaux et al, 2010;Bradbury and Pluckthun, 2015;Brooks and Lindsey, 2018) and reflected in policies for reagent validation that are now required by granting agencies (e.g. NIH).…”

Section: A Generalizable Workflow For Establishing Fit-for-purpose Usmentioning

confidence: 99%

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Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Waas

Weerasekera

Kropp

et al. 2018

Preprint

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Running Title: Standard flow cytometry protocol for assessing stem cell-derived cardiomyocytes SummaryModern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fitfor-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

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Section: Discussionsupporting

confidence: 66%

Section: A Generalizable Workflow For Establishing Fit-for-purpose Usmentioning

confidence: 99%

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Waas

Weerasekera

Kropp

et al. 2018

Preprint

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“…Immunoblotting was performed according to the Guidelines for authors and reviewers on antibody use in physiology studies [ 5 ]. Secretome (20 µl) was separated by 4–12% Criterion ™ XT Bis–Tris gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad).…”

Section: Materials and Methods (Detailed Methods In Supplemental Matementioning

confidence: 99%

Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis

et al. 2019

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Cardiac fibroblasts are the major producers of extracellular matrix (ECM) to form infarct scar. We hypothesized that fibroblasts undergo a spectrum of phenotype states over the course of myocardial infarction (MI) from early onset to scar formation. Fibroblasts were isolated from the infarct region of C57BL/6J male mice (3–6 months old, n = 60) at days 0 (no MI control) and 1, 3, or 7 after MI. Whole transcriptome analysis was performed by RNA-sequencing. Of the genes sequenced, 3371 were differentially expressed after MI. Enrichment analysis revealed that MI day 1 fibroblasts displayed pro-inflammatory, leukocyte-recruiting, pro-survival, and anti-migratory phenotype through Tnfrsf9 and CD137 signaling. MI day 3 fibroblasts had a proliferative, pro-fibrotic, and pro-angiogenic profile with elevated Il4ra signaling. MI day 7 fibroblasts showed an anti-angiogenic homeostatic-like myofibroblast profile and with a step-wise increase in Acta2 expression. MI day 7 fibroblasts relied on Pik3r3 signaling to mediate Tgfb1 effects and Fgfr2 to regulate PI3K signaling. In vitro, the day 3 MI fibroblast secretome stimulated angiogenesis, while day 7 MI fibroblast secretome repressed angiogenesis through Thbs1 signaling. Our results reveal novel mechanisms for fibroblasts in expressing pro-inflammatory molecules and regulating angiogenesis following MI.Electronic supplementary materialThe online version of this article (10.1007/s00395-019-0715-4) contains supplementary material, which is available to authorized users.

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“…Opal multiplex immunohistochemistry was performed on isolated neutrophils, using antibodies specific for nuclear factor (NF)κB-p65 or peroxisome proliferator-activated receptor (PPAR)γ, with DAPI as a nuclear counterstain and according to established guidelines for antibody use [5]. Images were acquired on the Mantra™ Quantitative Pathology Imaging microscope.…”

Section: Materials and Methods (Detailed Methods In Supplemental Matementioning

confidence: 99%

LXR/RXR signaling and neutrophil phenotype following myocardial infarction classify sex differences in remodeling

et al. 2018

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Sex differences in heart failure development following myocardial infarction (MI) are not fully understood. We hypothesized that differential MI signaling could explain variations in outcomes. Analysis of the mouse heart attack research tool 1.0 (422 mice; young = 5.4 ± 0.1; old = 23.3 ± 0.1 months of age) was used to dissect MI signaling pathways, which was validated in a new cohort of mice (4.8 ± 0.2 months of age); and substantiated in humans. Plasma collected at visit 2 from the MI subset of the Jackson Heart Study (JHS; a community-based study consisting of middle aged and older adults of African ancestry) underwent glycoproteomics grouped by outcome: (1) heart failure hospitalization after visit 2 (n = 3 men/12 women) and (2) without hospitalization through 2012 (n = 24 men/21 women). Compared to young male mice, the infarct region of young females had fewer, but more efficient tissue clearing neutrophils with reduced pro-inflammatory gene expression. Apolipoprotein (Apo) F, which acts upstream of the liver X receptors/retinoid X receptor (LXR/RXR) pathway, was elevated in the day 7 infarcts of old mice compared to young controls and was increased in both men and women with heart failure. In vitro, Apo F stimulated CD36 and peroxisome proliferator-activated receptor (PPAR)γ activation in male neutrophils to turn off NF-κB activation and stimulate LXR/RXR signaling to initiate resolution. Female neutrophils were desensitized to Apo F and instead relied on thrombospondin-1 stimulation of CD36 to upregulate AMP-activated protein kinase, resulting in an overall better wound healing strategy. With age, female mice were desensitized to LXR/RXR signaling, resulting in enhanced interleukin-6 activation, a finding replicated in the JHS community cohort. This is the first report to uncover sex differences in post-MI neutrophil signaling that yielded better outcomes in young females and worse outcomes with age.Electronic supplementary materialThe online version of this article (10.1007/s00395-018-0699-5) contains supplementary material, which is available to authorized users.

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Guidelines for authors and reviewers on antibody use in physiology studies

Cited by 71 publications

References 57 publications

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Fibroblast polarization over the myocardial infarction time continuum shifts roles from inflammation to angiogenesis

LXR/RXR signaling and neutrophil phenotype following myocardial infarction classify sex differences in remodeling

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