An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

Legroux, Laurine; Pittet, Camille L.; Beauseigle, Diane; Deblois, Gabrielle; Prat, Alexandre; Arbour, Nathalie

doi:10.1016/j.jneumeth.2015.03.021

Cited by 45 publications

(36 citation statements)

References 44 publications

(62 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were harvested and counted as described previously [15] with some modifications. Briefly, two retinas were minced in 1 ml of Hank’s Balanced Salt Solution (Thermo Fisher Scientific) containing 2 mg/ml Collagenase D and 28 U/ml DNase I (both from Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130

Wang

Miller

Goswami

et al. 2017

J Neuroinflammation

View full text Add to dashboard Cite

BackgroundRetinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice.MethodsRD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor β (gp-130) was injected intravitreally immediately after RD.ResultsWithin one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade.ConclusionsTogether, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0886-6) contains supplementary material, which is available to authorized users.

show abstract

Section: Methodsmentioning

confidence: 99%

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130

Wang

Miller

Goswami

et al. 2017

J Neuroinflammation

View full text Add to dashboard Cite

show abstract

“…Microglia and brain leukocytes were analyzed using and adaptation of the protocol described by Legroux et al (Legroux et al, 2015). Briefly, mice were perfused with icecold PBS containing 10 U/ml heparin.…”

Section: Analysis Of Microglia and Leukocytesmentioning

confidence: 99%

Microglial homeostasis requires balanced CSF-1/CSF-2 receptor signaling

Chiţu

Biundo

Shlager

et al. 2019

Preprint

View full text Add to dashboard Cite

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r +/mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r +/mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in man. Our data provide new insights into the mechanisms underlying ALSP. Since both increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions. Abbreviations: CSF-1 RColony stimulating factor-1 receptor CSF-2Colony stimulating factor-2 ALSP Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia Highlights• ALSP is a CSF1R-deficiency dementia associated with increased CSF2 expression • In Csf1r+/-ALSP mice CSF-2 promotes microgliosis by direct signaling in microglia • Targeting Csf2 improves cognition, myelination and normalizes microglial function • CSF-2 is a therapeutic target in ALSP

show abstract

“…The collagenase based approach has been employed by our laboratory as well as others as a standard method to isolate brain-infiltrating lymphocytes [8, 16, 17, 24–29]. Manual homogenization has also been employed among various research laboratories as a method of brain-infiltrating lymphocyte isolation as well [15, 30, 31].…”

Section: Discussionmentioning

confidence: 99%

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique

Garcia

Kelcher

Malo

et al. 2016

Journal of Immunological Methods

View full text Add to dashboard Cite

Effective recovery of activated brain infiltrating lymphocytes is critical for investigations involving murine neurological disease models. To optimize lymphocyte recovery, we compared two isolation methods using brains harvested from seven-day Theiler’s murine encephalomyelitis virus (TMEV) and TMEV-OVA infected mice. Brains were processed using either a manual dounce based approach or enzymatic digestion using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62lo activated lymphocytes and CD19+ B cells using the dounce method. In addition, we achieved a 3-fold greater recovery of activated virus-specific CD8 T cells specific for the immunodominant Db:VP2121-130 and engineered Kb:OVA257-264 epitopes through manual dounce homogenization approach as compared to collagenase digest. A greater percentage of viable cells was also achieved through dounce homogenization. Therefore, we conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.

show abstract

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

Cited by 45 publications

References 44 publications

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130

Microglial homeostasis requires balanced CSF-1/CSF-2 receptor signaling

Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique

Contact Info

Product

Resources

About