Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: Immunostaining after Recording ATP-elicited Ca 2+ Signals in pancreatic macrophages (PMs). (A). Representative images of pancreatic lobule loaded with Fluo-4AM before (Ai) and after ATP (10 µM) application (Aii), the arrow indicates the position of the ( n = 8). A corresponding Fluo 4 trace from a PM is shown in Aiii . Corresponding immunostaining of this lobule with antibodies F4/80 Alexa Fluo 647 is shown below ( Aiv) . Hoechst 33342 staining of the same area is shown in Av . Arrow points to ear-like shape of PM nucleus. Overlay of antibody and Hoechst 33342 staining is shown in Avi . Scale bar is 10µm. (B). Immunostaining of another area in a pancreatic lobule with monoclonal F4/80 antibodies labeled with Alexa Fluor 647 (Bi) . Staining of nuclei in the same lobule with Hoechst 33342 (Bii) . Overlay of B i with Bii is shown in Biii . Scale bar is 10µm. (C). Representative images of a pancreatic lobule loaded with Fluo-4AM before (Ci) and after ATP (10 µM) application (Cii) , the arrow indicates the position of the PM. Corresponding Fluo 4 trace is shown in Ciii. Immunostaining of the same area with monoclonal CD11b antibody conjugated with Alexa Fluor 647 ( n = 8) is shown in Civ . Overlay of Cii and Civ is shown in Cv . Scale bar is 10µm.

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Immunostaining, Staining, Labeling

Journal: Function

Article Title: Calcium Signaling in Pancreatic Immune Cells In situ

doi: 10.1093/function/zqaa026

Figure Lengend Snippet: IgG-elicited Ca 2+ Spikes in PMs . (A). Single short Ca 2+ spike occurring after application of IgG (0.1–0.25 mg/mL) in a PM from a control pancreatic lobule. This was an infrequent observation (5 out of 29 cells tested) and is most likely not an IgG-elicited Ca 2+ signal as such single spikes have been also observed in 3 out of 15 cells in the absence of IgG stimulation. (B) . Representative trace of IgG (0.1–0.25 mg/mL)-induced Ca 2+ signals in PMs in pancreatic lobules isolated from mice with AP (FAEE-AP model—48 h). Such oscillations were observed in 9 out of 31 cells. Single short spikes have been observed in 4 out of 31 cells. No oscillations were observed in the absence of stimulation with IgG ( n = 14), while single short spikes have been observed in 2 out of 14 cells. (C). Average Ca 2+ spike frequencies in PMs displaying Ca 2+ signals under the conditions indicated. The frequencies in control PMs, both stimulated with IgG (blue bar) and unstimulated (green), as well as in unstimulated PMs from the FAEE-AP model (48 h, orange bar) were much lower than in PMs from the FAEE-AP model stimulated with IgG (red bar, P < 0.007). (D) . Average Ca 2+ spike duration in PMs displaying Ca 2+ signals under the conditions indicated. Although the average spike duration was longer in the PMs from the FAEE-AP mice stimulated with IgG than under the other conditions, the difference was not statistically different ( P > 0.2). (E). Representative images of immunostaining of PMs in lobules using antibodies F4/80 conjugated with Alexa Fluor 647. Lobules were isolated from control and FAEE-AP 3-day mice (72 h in vivo FAEE-AP model). Scale bar is 20µm. (F). Comparison of the average density of PMs in lobules from control and FAEE-AP 2-day and 3-day mice (48 h and 72 h in vivo FAEE-AP model, respectively). Control, 2.36 ± 0.6 SEM, n = 14; FAEE-AP 2 day, 9.56 ± 1.86 SEM, * P < 0.033, n = 16; FAEE-AP 3 days, 15.37 ± 1.51 SEM, * P < 0.038 as compared to FAEE-AP 2-day, n = 35. The difference between control and FAEE-AP 3-day was very highly significant (**** P < 0.0001).

Article Snippet: Mouse F4/80 monoclonal rat Antibody (CI-A3-1) [Alexa Fluor ® 647] and mouse CD11b/Integrin alpha M Alexa Fluor ® 647-conjugated monoclonal rat antibodies were obtained from Novus Biologicals Europe and R&D Systems Bio-techne, respectively.

Techniques: Control, Isolation, Immunostaining, In Vivo, Comparison

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 μg/10 6 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation

Journal: Cell Reports

Article Title: Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity

doi: 10.1016/j.celrep.2018.08.006

Figure Lengend Snippet:

Article Snippet: The human/mouse Anti-UCP1 antibody (MAB6158, monoclonal Mouse IgG 2B Clone # 536435, R&D Systems) was conjugated to Alexa647 (ThermoFisher Alexa Fluor 647 antibody labeling kit A20186) and incubated with adipocytes at 1:300 for 1 h. Mouse IgG2B Alexa Fluor 647-conjugated Isotype Control antibody (R&D systems) was used as a control.

Techniques: Control, Recombinant, Blocking Assay, Antibody Labeling, Software, Flow Cytometry

Journal: Cell reports

Article Title: Pericyte dysfunction and impaired vasomotion are hallmarks of islets during the pathogenesis of type 1 diabetes

doi: 10.1016/j.celrep.2023.112913

Figure Lengend Snippet: Living pancreas slices were incubated with Fluo4 and NG2-Alexa647. (A) Confocal image of a pericyte in an islet from a NDdonor (nPOD6516) in basal glucose concentration (3 mM), upon high glucose (11 mM) alone, or in the presence of theophylline (20 μM; 11G + theo). Scale bar: 10 μm. (B) Heatmap showing changes in Fluo4 fluorescence (DF/F; %) in pericytes in islets from ND donors elicited by 11 mM glucose and then theo (20 μM; in 11G). Each line is a different pericyte. (C) Representative traces showing changes in Fluo4 fluorescence elicited by glucose and theo. (D) Net area under the curve (AUC) of fluorescence traces for individual pericytes. *p < 0.05 (paired t test; n = 113 pericytes from 5 ND donors). (E) Heatmaps showing changes in Fluo4 fluorescence in pericytes from ND (n = 9), Aab+ (n = 7), and T1D donors (n = 6). Donors are separated by white dashed lines. T1D donor data are presented based on T1D duration, while data from ND and Aab+ donors are presented according to . (F) Traces showing average changes in Fluo4 fluorescence elicited 11mM glucose for all islet pericytes from each group. Average (thicker lines) ± SEM values are shown. (G) Net AUC of fluorescence traces during stimulation with 11G (dots: data point for each donor, bars: mean ± SEM). For each donor, responses from around 3–36 pericytes were recorded, and an average value calculated. *p < 0.05 (one-sample t test, theoretical mean = 0).

Article Snippet: Living human pancreas slices were incubated with the cytosolic calcium indicator ([Ca 2+ ]i) Fluo4-AM (6 mM, Invitrogen, cat. nr. F14201) in 3 mM glucose solution prepared in HEPES buffer (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl 2 , 1 mM MgCl2, 25 mM HEPES, 0.1% BSA [w/v], pH 7.4), supplemented with aprotinin (25 KIU), at room temperature and in the dark with either (a) DyLight 649 lectin from Lycopersicon Esculentum (3.3 mg/mL, VectorLabs, cat. nr. DL1178) for 1 hour; or with (b) a fluorescent-conjugated antibody against the pericyte marker neuron-glial antigen 2 (NG2-alexa647; 1:50, R&D Systems, cat. nr. Fab2585R) for 2 hours.

Techniques: Incubation, Concentration Assay, Fluorescence