Search

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

Cadmium (Cd) is a widespread toxic occupational and environmental pollutant, and its effect on lipid metabolism dysregulation has been observed. In this study, we utilized two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) technologies to explore changes in the blood plasma proteins of mice exposed to Cd. From the 2-DE, 8 protein spots were screened in response to Cd exposure, and the identities of these proteins were revealed by MALDI-TOF MS. Western blotting was applied to analyze the expression of the apolipoproteins in both plasma and liver, which were consistent with Cd-induced dyslipidemia of their composed lipid. Moreover, the Cd-induced apolipoprotein ApoE upregulation was due to inhibition of autopahgic flux in the Cd exposed mice. It was further observed from the mouse liver that Cd reduced the expression of the lipid uptake receptor low-density lipoprotein receptor (LDLR), which might be responsible for the coordinated elevation in blood triglycerides and abnormal apolipoproteins. This study may provide a new insight into the mechanism of Cd-induced dyslipidemia and the risk of cardiovascular diseases.

Significance There continues to be a paucity in models to target mononuclear phagocyte (MP) subsets selectively and in specific tissues. We report a mouse model allowing for selective depletion of a specific subset of kidney MPs that is based on a Cre-inducible lox-STOP-lox-diphtheria toxin receptor gene controlled by the endogenous CD64 promoter. Combined with specific targeting of conventional DC1 (cDC1) and cDC2, we revealed that CD64+ MPs account for the reported ability of CD11c+ cells to limit cisplatin nephrotoxicity, while cDC1 and cDC2 are dispensable. Our study highlights that individual MP subsets have unique functions in kidney pathology. Combined with various CRE drivers, CD64-lox-STOP-lox-diphtheria toxin receptor mice might be used to study CD64-expressing cells in other tissues., Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin–mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c+ cells to limit cisplatin toxicity is specifically attributed to CD64+ MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression.

Human exposure to cadmium (Cd) could lead to alterations in lipid metabolism. However, the underlying mechanism is still unclear. In the present study, the data revealed that Cd exposure induced cholesterol redistribution both in vivo from mouse liver tissue into the serum, and in vitro from the HepG2 cells to the cultured medium, which were associated with modulating the expressions of cholesterol efflux proteins, including upregulating cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) and downregulating oxysterol-binding protein (OSBP). Further investigation in HepG2 cells revealed that Cd upregulated ABCA1 expression with increased stability by inhibiting lysosomal pathway, and downregulated OSBP expression by increasing ubiquitination. Cd-induced cholesterol redistribution was completely inhibited by knockdown of ABCA1 expression using siRNA, and was significantly reduced by overexpression of OSBP. Taken together, these results suggested that Cd induced cholesterol redistribution by upregulating ABCA1 and downregulating OSBP.

In this work, the synthesis of regular single crystalline lead iodide nanoplatelets are carried out based on the physical vapor phase deposition method. Different lead iodide nanoplatelets are obtained by tuning the location of the mica substrate along with the temperature of the tube furnace. The rules of size, thickness, density of the lead iodide nanoplatelets at varied deposition conditions are analyzed according to the crystal growth principles. It was claimed in literature that the photoluminescence of lead iodide could be obtained only at a low temperature (lower than 200 K). Here, at room temperature, we successfully obtained the photoluminescence spectra of the prepared lead iodide nanoplatelets, which possess two apparent peaks due to the biexcitons and the inelastic scattering of excitons, respectively. Our present study contributes to the development of nanoscaled high performance optoelectronic devices.

Additional file 1: Figure S1. SDS-PAGE analysis of BglucH and BglucLEH enzyme. (a): SDS–PAGE analysis of BglucH enzyme and BglucLEH enzyme induce at 37 °C. Lane M, Protein molecular weight marker (Broad); Lane 1, cell lysates of BglucH induced 0 h; Lane 2, cell lysates of BglucH induced 8 h; Lane 3, precipitation lysates containing BglucH; Lane 4, crude cell lysate of BglucH; Lane 5, crude cell lysate of empty pET-28a(+) vector; Lane 6, cell lysates of BglucLEH induced 0 h; Lane 7, cell lysates of BglucLEH induced 8 h; Lane 8: precipitation lysates containing BglucLEH; Lane 9, crude cell lysate of BglucH; The arrows in the figure are the insoluble fractions of the BglucH enzyme and the BglucLEH enzyme. (b): SDS–PAGE analysis of BglucH enzyme and BglucLEH enzyme induced at 25 °C. Lane M, Protein molecular weight marker (Broad); Lane 1, cell lysates of BglucH induced 0 h; Lane 2, cell lysates of BglucH induced 8 h; Lane 3, precipitation lysates containing BglucH; Lane 4, crude cell lysate of BglucH; Lane 5, crude cell lysate of empty pET-28a(+) vector; Lane 6, cell lysates of BglucLEH induced 0 h; Lane 7, cell lysates of BglucLEH induced 8 h; Lane 8: precipitation lysates containing BglucLEH; Lane 9, crude cell lysate of BglucH; The red wireframe in the figure indicates the whole bacteria, sediment and supernatant solution containing BglucH enzyme and BglucLEH enzyme, respectively. Figure S2. SDS–PAGE analysis of BglucH induced at 25 °C and purification by Ni-NTA resin. Lane M, Protein molecular weight marker (Broad); Lane 1, crude cell lysate of cell with empty pET-28a(+) vector; Lane 2, crude cell lysate containing BglucH; Lanes 3–4, purified BglucH by Ni-NTA resin. Figure S3. SDS–PAGE analysis of twice ITC and 400mM imidazole purified BglucLEH. Lane M, Protein molecular weight marker (Broad); Lane 1, crude cell lysate of cell with empty pET-28a(+) vector; Lane 2, crude cell lysate containing BglucLEH; Lanes 3–4, BglucLEH purified after one and two rounds of ITC operation using 0.5 M of (NH4)2SO4. Figure S4. Lineweaver–Burk plots for (a) BglucLEH, (b) BglucLEH.

The issue of organic-inorganic perovskites has received considerable attention because of its importance in improving the efficiency of solar cells. The power conversion efficiency of solar cells has climbed from 3.8% in 2009 to over 25.2% currently. Its outstanding performance is mainly attributed to its long carrier lifetime (101−2 ns), diffusion length (μm), high absorption coefficient, direct bandgap (band gap ~1.55 eV), stable electron/hole migration and other characteristics. In addition to these features, organic-inorganic lead perovskites also have high fluorescence yields and tunable absorption wavelengths, making it an ideal laser gain medium. In this paper, the physical vapor deposition (PVD) is used to grow atomically smooth lead iodide nanoplatelets (PbI2 NPL) on a mica substrate, and convert the PbI2 NPL into atomically smooth single-crystal perovskite nanoplatelets (CH3NH3PbI3 NPL) by chemical vapor deposition. The nanoplatelets can naturally form WGM (whispering-gallery-mode) microcavities. We can control the size of CH3NH3PbI3 NPL (from a few microns to dozens of microns) by adjusting the deposition location, deposition temperature, and deposition time. With the increase of edge length, the mode interval decreases, the peak is red shift, and it shows excellent lasing characteristics with a threshold down to 17 μJ/cm2. This has great application potential in the field of low-cost chip light source.

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.

Cadmium (Cd), as an extremely toxic metal could accumulate in kidney and induce renal injury. Previous studies have proved that Cd impact on renal cell proliferation, autophagy and apoptosis, but the detoxification drugs and the functional mechanism are still in study. In this study, we used mouse renal tubular epithelial cells (mRTECs) to clarify Cd-induced toxicity and signaling pathways. Moreover, we proposed to elucidate the prevent effect of activation of Ca2+ sensing receptor (CaSR) by Calcimimetic (R-467) on Cd-induced cytotoxicity and underlying mechanisms. Cd induced intracellular Ca2+ elevation through phospholipase C-inositol 1, 4, 5-trisphosphate (PLC) followed stimulating p38 mitogen-activated protein kinases (MAPK) activation and suppressing extracellular signal-regulated kinase (ERK) activation, which leaded to increase apoptotic cell death and inhibit cell proliferation. Cd induced p38 activation also contribute to autophagic flux inhibition that aggravated Cd induced apoptosis. R-467 reinstated Cd-induced elevation of intracellular Ca2+ and apoptosis, and it also increased cell proliferation and restored autophagic flux by switching p38 to ERK pathway. The identification of the activation of CaSR-mediated protective pathway in renal cells sheds light on a possible cellular protective mechanism against Cd-induced kidney injury.