anti acsa 2 conjugated microbeads  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: Culture density influences the functional phenotype of human macrophages

doi: 10.3389/fimmu.2023.1078591

Figure Lengend Snippet: Density affects human primary MΦ function. CD14-positive monocytes were isolated from PBMCs of 6 healthy donors, pooled and seeded at the respective densities (1-6 x 10 3 cells/mm²) and differentiated to MΦ using 100 ng/ml macrophage colony-stimulating factor (M-CSF) for 7 days. Phagocytic activity (A , red ) , oxLDL uptake (B , green ) , inflammasome activation (C , red ) , and mitochondrial stress (D , red ) were assessed using the MacroScreen platform. Hoechst was used for nuclear labelling (blue). The scale bar in the representative fluorescent images refers to 50 μM. The indicated p-values refer to overall significance, multiple comparison significance can be found in Supplementary Table 2 . MFI: Mean fluorescence intensity.

Article Snippet: CD14 MicroBeads (Miltenyi #130-096-052) were used to positively select CD14+ monocytes, following the manufacturer’s protocol.

Techniques: Isolation, Activity Assay, Activation Assay, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: Culture density influences the functional phenotype of human macrophages

doi: 10.3389/fimmu.2023.1078591

Figure Lengend Snippet: MΦ function differs between donors. CD14-positive monocytes of 6 healthy donors were seeded at 13,000 cells/well and differentiated into MΦ for 7 days using 100 ng/ml macrophage colony-stimulating factor (M-CSF). The number of nuclei per donor at experimentation (A) is given. Their functional phenotype was assessed using the MacroScreen platform: phagocytosis (B , red ) , lipid uptake (C , green ) , inflammasome activation (D , red ) , and mitochondrial stress (E , red ) . The scale bar corresponds to 25 μM. The indicated p-values refer to overall significance, multiple comparison significance can be found in Supplementary Table 3 . MFI: Mean fluorescence intensity.

Article Snippet: CD14 MicroBeads (Miltenyi #130-096-052) were used to positively select CD14+ monocytes, following the manufacturer’s protocol.

Techniques: Functional Assay, Activation Assay, Comparison, Fluorescence

Journal: Croatian Medical Journal

Article Title: Uroguanylin increases Ca 2+ concentration in astrocytes via guanylate cyclase C-independent signaling pathway

doi: 10.3325/cmj.2021.62.250

Figure Lengend Snippet: Uroguanylin (UGN, 100 nM) changes the H + and HCO 3 - transport in primary astrocyte cultures. UGN increases the Na + /H + exchanger activity measured by ammonia pulse ( A ) and HCO 3 - transport ( B ); summarized effects ( C ). The mechanism of cytoplasm alkalinization due to the activation of Na + /H + exchanger and HCO 3 - transport is presented. The mean of the slopes of the control experiments was set to 100%. The results are shown as mean ± standard deviation (SD), n = 3-5, primary culture isolated from three newborn animals. Bar represents 100 s. Asterisk indicates significant difference compared with controls, P < 0.05. Continuous line – control; dashed line – UGN.

Article Snippet: Supernatant-containing cells were collected and centrifuged for 6 min at 300 g. The supernatant solution was removed, and cell pellet was subjected to magnetic activated cell separation, as previously described (Astrocytes Isolation Starter Kit, Cat. No. 130-096-054, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) ( ).

Techniques: Activity Assay, Activation Assay, Control, Standard Deviation, Isolation

Journal: Croatian Medical Journal

Article Title: Uroguanylin increases Ca 2+ concentration in astrocytes via guanylate cyclase C-independent signaling pathway

doi: 10.3325/cmj.2021.62.250

Figure Lengend Snippet: Guanylate cyclase C (GC-C) is expressed in neurons of different brain regions. GC-C (green) co-localized with a neuronal nuclei marker (NeuN = red, left panel) but not with the astrocytes marker, glial fibrillary acidic protein (GFAP = red, right panel) in ( A ) the hypothalamus, ( B ) cerebellar cortex, ( C ) cerebellar deep nuclei, and ( D ) cerebral cortex of wild-type mice. GC-C knockout (KO) mice were used as a negative control. Bar represents 50 μm.

Article Snippet: Supernatant-containing cells were collected and centrifuged for 6 min at 300 g. The supernatant solution was removed, and cell pellet was subjected to magnetic activated cell separation, as previously described (Astrocytes Isolation Starter Kit, Cat. No. 130-096-054, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) ( ).

Techniques: Marker, Knock-Out, Negative Control

Journal: Croatian Medical Journal

Article Title: Uroguanylin increases Ca 2+ concentration in astrocytes via guanylate cyclase C-independent signaling pathway

doi: 10.3325/cmj.2021.62.250

Figure Lengend Snippet: Uroguanylin (UGN) Ca 2+ signaling pathway in the cortical astrocytes of brain slices. mRNA of guanylate cyclase C (GC-C) (341 bp) is expressed in the hypothalamus (H), midbrain (MB), cerebral cortex (Cx), and cerebellum (Cb) but not in astrocytes (A) ( A ). M – marker, In – intestine as positive control, Neg – negative control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 235 bp) was used as cDNA control. Ca 2+ measurements were performed only in SR101-positive cells ( B ) of GC-C knockout (KO) (left, the number of animals is 3, the number of brain slices is 6, the number of cells is 32) and UGN KO (right, the number of animals is 3, the number of brain slices is 5, the number of cells is 14) cerebral cortex, and the results are compared with those of their wild-type littermates (GC-C WT: the number of animals is 4, the number of brain slices is 6, the number of cells is 28; UGN WT: the number of animals is 3, the number of brain slices is 5, the number of cells is 17). In neurons of the cerebral cortex of WT animals, UGN did not affect the intracellular Ca 2+ concentration (the number of animals is 4, the number of brain slices is 8, the number of cells is 43). Hyperkalemia of 30 mM was used as a positive control ( C ). The results are shown as mean ± standard deviation (SD) ΔF/F 0 is change of light output in time (ΔF) over initial brightness (F 0 ). Bar represents 10 s.

Article Snippet: Supernatant-containing cells were collected and centrifuged for 6 min at 300 g. The supernatant solution was removed, and cell pellet was subjected to magnetic activated cell separation, as previously described (Astrocytes Isolation Starter Kit, Cat. No. 130-096-054, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) ( ).

Techniques: Marker, Positive Control, Negative Control, Control, Knock-Out, Concentration Assay, Standard Deviation

Journal: Croatian Medical Journal

Article Title: Uroguanylin increases Ca 2+ concentration in astrocytes via guanylate cyclase C-independent signaling pathway

doi: 10.3325/cmj.2021.62.250

Figure Lengend Snippet: Uroguanylin (UGN) Ca 2+ signaling pathway in primary astrocyte cultures. An electrophysiological recording showing an effect of UGN (10 nM) opposite to that of membrane permeable cyclic guanosine monophosphate (cGMP, 100 μM) on astrocyte membrane potential – original trace. Dashed line represents starting membrane potential ( A ). Guanylin (GN, 10 nM) and UGN hyperpolarized, while membrane permeable cGMP depolarized astrocytes ( B ). Hyperpolarization caused by guanylin peptides was negatively correlated with the starting membrane potential ( C ). UGN (100 nM) and bradykinin (BK, 1 μM – positive control) increased intracellular Ca 2+ concentration ( D ). The results are shown as mean ± standard deviation (SD). The number of experiments is shown in parentheses. Octothorpe indicates a significant difference between cGMP effects and effects of guanylin peptides, at P < 0.05. Asterisk indicates a significant difference between UGN and BK effects, at P < 0.05. ΔF/F 0 is change of light output in time (ΔF) over initial brightness (F 0 ). Bar represents 10 s.

Article Snippet: Supernatant-containing cells were collected and centrifuged for 6 min at 300 g. The supernatant solution was removed, and cell pellet was subjected to magnetic activated cell separation, as previously described (Astrocytes Isolation Starter Kit, Cat. No. 130-096-054, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) ( ).

Techniques: Membrane, Positive Control, Concentration Assay, Standard Deviation