Journal: Cell metabolism

Article Title: Multiplexed in Situ Imaging Mass Cytometry Analysis of the Human Endocrine Pancreas and Immune System in Type 1 Diabetes

doi: 10.1016/j.cmet.2019.01.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 159Tb-CD68 (Clone KP1) , Fluidigm , Cat#3159035D.

Techniques: Software

Journal: Current protocols

Article Title: Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro.

doi: 10.1002/cpz1.70107

Figure Lengend Snippet: Figure 15 THP-1 dramatic polarization toward an M1-like phenotype with Sepiapterin treatment in the 3D Flipwell coculture system.(A) Confocal images of THP-1 cells cocultured with Caco-2:HT- 29 in the 3D Flipwells and stained for pan macrophage marker CD68 (red) and CD80 (green). Pan macrophage CD68 marker is present in both the SEP treated sample and the untreated control. CD80 marker, typical of the M1 polarization phenotype, is more prominently present in the SEP treated sample with long pseudopodia extending outward typical of the M1 phenotype. (B) Con- focal images of THP-1 cells cocultured with Caco-2:HT-29 in the 3D Flipwell and stained for pan macrophage marker CD68 and CD163 to validate phenotypic polarization toward M1 phenotype and away from M2 phenotype. Scale bar = 25 μm.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Anti-human MUC2 (996/1) (Thermo Fisher, cat. no. MA5-12345) Anti-human CK20 (Life Technologies, cat. no. PA5-82875) Anti-human CD80 (Cell Signaling Technologies, E3Q9V, cat. no. 15416) Anti-human CD163 (Abcam, cat. no. 156769) or anti-human CD68 (Novus Biologicals, cat. no. NB100-683) Secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (Thermo Fisher, cat. no. A11008) for CD80 and CK20 Alexa Fluor 594 goat anti-mouse IgG (Thermo Fisher, cat. no. A11005) for MUC2 and CD68 or CD163 DAPI, VectaShield mounting medium (Vector Laboratories, cat. no. H-1000) 50-ml conical tubes (SpectraTube, Alkali Scientific cat. no. C2750) Plastic wrap Lab tape or shipping tape Tweezers 12-well tissue culture plate (Corning Costar, cat. no. 3513) 6-well dish Aluminum foil Scalpel Round cover slips, #1, 1 oz (Corning Float Glass, cat. no. 50143822, or Fisherbrand cat. no. 12-546-2 25CIR-2) Laboratory marker (VWR, cat. no 95042-566) Attofluor cell chamber coverslip holder (Thermo Fisher Scientific, cat. no. A7816) Confocal microscope and software [e.g., Leica Microsystems TCS SP5, multi-photon laser scanning, and Suite Advanced Fluorescence (LAS AF) software] Deep Petri dish bottoms, 100 × 25–mm deep polystyrene stackable Petri dish, sterile (USA Scientific, cat. no. 8609-0625) 10-, 200-, and 1000-μl pipet tips (Alkali Scientific PurePoint or equivalent) Beckman Coulter Allegra 6 centrifuge with a swing bucket rotor for 50-ml conical tubes Optional: ImmunoPen (Fisher Scientific, cat. no. 4021761EA) Wash, permeabilization, disassembly, and blocking steps 1.

Techniques: Staining, Marker, Control

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Representative photos showing hind paw of rat receiving CPIP model establishment. ( B ) Time course showing the thickness of ipsilateral hind paw of sham and CPIP model rats. n = 5 rats per group. ( C ) Time course showing 50% paw withdrawal threshold (PWT) from sham and CPIP model rats. n = 5 to 6 rats per group. ( D ) Flow chart of scRNA-seq. Created in BioRender. Pan, Y. (2025) https://BioRender.com/wd2ghrz . ( E ) UMAP plot of cell clusters identified by scRNA-seq. vSMC, vascular smooth muscle cell. ( F ) Cell populations identified from immune cell cluster by marker genes. ( G ) Distribution of specific immune cell population (including macrophages, neutrophils, and T or B cells) in DRG of sham versus CPIP model rats. ( H ) Summary of increase in the number of cells in DRG after CPIP modeling. ( I ) CD68 (red, marker for macrophage) and NeuN (green, marker for neuron) immunostaining in ipsilateral L4-L6 DRG from sham and CPIP groups. Scale bar, 50 μm. ( J ) Flow cytometry of macrophages expressing CD68 in cell suspensions from DRG of sham and CPIP groups on day 7. A total of 10,000 cells were analyzed for each. ( K ) Summary of the percentage of CD68 + cells in DRG as in (J). n = 3 to 4 rats per group. ( L ) Representative pictures showing CD68 immunostaining in ipsilateral L4-L6 DRG 3, 7, and 14 days after model establishment. 4′,6-Diamidino-2-phenylindole (DAPI) was used as counterstain. Scale bar, 50 μm. ( M ) Summary of the number of CD68 + cells/mm 2 as in (L). n = 5 to 6 rats per group. 7D, 7 days. ( N ) Representative pictures showing CD68 immunostaining in ipsilateral sciatic nerve. Scale bar, 100 μm. ( O ) Summary of the number of CD68 + cells/mm 2 as calculated in (N). n = 6 rats per group. * P < 0.05 and ** P < 0.01 versus sham group. NS, no significance. Two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for [(B) and (C)]. One-way ANOVA followed by Tukey’s post hoc test for (M). Student’s unpaired t test for [(K) and (O)].

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Marker, Immunostaining, Flow Cytometry, Expressing

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Time schedule. ( B ) CD68 immunostaining in ipsilateral L4-L6 DRG of sham + IgG, CPIP + IgG, and CPIP + Reg3β-neutralizing Ab groups. Scale bar, 50 μm. ( C ) Summary of the number of CD68 + cells/mm 2 from three groups as in (B). n = 7 rats per group. ( D ) 50% PWT changes in three groups. n = 8 to 10 rats per group. * P < 0.05 and ** P < 0.01 versus sham + IgG group. ## P < 0.01 versus CPIP + IgG group. ( E ) Strategy for Reg3b -specific knockdown in DRG neurons using AAV-PHP.S with neuronal promoter hSyn. ( F ) EGFP detected in DRG of control (without viral injection), scramble-transfected, and Reg3b shRNA-transfected groups. Scale bar, 100 μm. ( G ) Summary of the percentage of EGFP + NeuN + cells in DRG of three groups as in (F). n = 6 rats per group. ( H ) Western blotting showing Reg3β expression in DRG of CPIP + scramble versus CPIP + Reg3b shRNA group. n = 5 rats per group. ( I ) CD68 + immunostaining in DRG of sham + scramble, CPIP + scramble, and CPIP + Reg3b shRNA groups. Scale bar, 50 μm. ( J ) Summary of the number of CD68 + cells in DRG of three groups of rats as in (I). n = 5 rats per group. ( K ) 50% PWT changes in sham + scramble, CPIP + scramble, and CPIP + Reg3b shRNA groups. n = 5 to 6 rats per group. ** P < 0.01 versus sham + scramble group. # P < 0.05 versus CPIP + scramble group. ( L ) Area under the curve (AUC) analysis of the curves shown in (K). n = 5 to 6 rats per group. ( M ) C-Fos immunostaining in ipsilateral SCDH of three groups. C-Fos was magnified and shown in the upper right, and its outline was illustrated in lower right. Scale bar, 50 μm. ( N ) Summary of the number of c-Fos + cells in ipsilateral SCDH per observation field as shown in (M). n = 5 to 6 rats per group. * P < 0.05 and ** P < 0.01. One-way ANOVA followed by Tukey’s post hoc test for [(C), (G), (J), (L), and (N)]. Student’s unpaired t test for (H). Two-way ANOVA followed by Tukey’s post hoc test for [(D) and (K)]. w, weeks; d, days.

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Immunostaining, Knockdown, Control, Injection, Transfection, shRNA, Western Blot, Expressing

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Strategy for Reg3b -specific overexpression in naïve rat DRG neurons using AAV-PHP.S with neuronal promoter hSyn. i.pl, intraplantar. ( B ) Time points for experiments in this study. ( C ) EGFP detected in ipsilateral DRG of control (nontransfected) and AAV-PHP.S-hSyn-EGFP (AAV-EGFP)–transfected rats. Scale bar, 100 μm. ( D ) Summary of the percentage of EGFP + NeuN + cells in DRG as shown in (C). n = 5 rats per group. ( E ) Western blotting examining Reg3β expression in DRG of AAV-EGFP versus AAV-PHP.S-hSyn- Reg3b -EGFP (AAV- Reg3b -EGFP) groups of rats. n = 6 rats per group. ( F ) EGFP signals in ipsilateral/contralateral SCDH of control and AAV-EGFP–transfected naïve rats. Scale bar, 100 μm. ( G and H ) Summary of the normalized fluorescence intensity of EGFP in ipsilateral/contralateral SCDH of control and AAV-EGFP–transfected naïve rats as shown in (F). n = 5 to 6 rats per group. ( I ) 50% PWT changes in the injected hind paw (ipsilateral hind paw) of naïve rats transfected with AAV-EGFP or AAV- Reg3b -EGFP. n = 5 rats per group ** P < 0.01 versus AAV-EGFP group. ( J ) Immunostaining of CD68 + cells (in red) in ipsilateral DRG of rats receiving AAV-EGFP or AAV- Reg3b -EGFP transfection. DAPI is in purple. Scale bar, 50 μm. ( K ) Summary of the number of CD68 + cells/mm 2 in DRG of two groups of rats as shown in (J). n = 5 to 6 rats per group. ** P < 0.01. Student’s unpaired t test for [(D), (E), (G), (H), and (K)]. Two-way ANOVA followed by Tukey’s post hoc test for (I).

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Over Expression, Control, Transfection, Western Blot, Expressing, Fluorescence, Injection, Immunostaining

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) KEGG analysis of signaling pathways activated in macrophages in DRG of CPIP rats. ( B ) Tnfa expression by qPCR in DRG. n = 5 rats per group. ( C ) Tnfa , Il6 , Il1b , and Cxcl1 expression in DRG of sham, CPIP + Lipo, and CPIP + Clodro groups. n = 4 to 5 rats per group. ( D ) Tnfa (red) expression in ipsilateral DRG by RNAscope. DAPI was in white. Negative control probe against bacterial gene Dapb shows no staining. ( E ) Summary of Tnfa + signals in DRG as in (D). n = 5 rats per group. ( F ) Colocalization of Cd68 (green) with Tnfa (red) in DRG of CPIP rats by RNAscope. ( G ) Summary of Tnfa expression in certain types of cells in DRG of CPIP rats by scRNA-seq. NK, natural killer. ( H ) Schedule. ( I ) 50% PWT in ipsilateral hind paws of sham + Veh, CPIP + Veh, and CPIP + ETA groups. ** P < 0.01 versus sham group. ## P < 0.01 versus CPIP + Veh group. ( J ) Normalized AUC analysis of curves in (I). n = 6 rats per group. ( K ) APs in small-sized DRG neurons of three groups. Five hundred–picoampere depolarizing current (1000 ms) was used to stimulate APs firing. ( L ) Number of APs elicited by 500-pA depolarizing current as in (K). n = 23 to 25 cells per group. ( M ) Summary of the number of APs triggered by depolarizing current steps in DRG neurons. n = 23 to 25 cells per group. * P < 0.05 and ** P < 0.01 versus sham + Veh group. # P < 0.05 and ## P < 0.01 versus CPIP + Veh group. ( N ) APs elicited by ramp current. ( O ) Summary of the number of APs triggered by ramp current. n = 23 to 27 cells per group. ( P ) Summary of current threshold for eliciting APs by ramp current. n = 23 to 27 cells per group. One-way ANOVA followed by Tukey’s post hoc test for [(B), (C), (E), (J), (L), (O), and (P)]. Two-way ANOVA followed by Tukey’s post hoc test for [(I) and (M)].

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Protein-Protein interactions, Expressing, RNAscope, Negative Control, Staining

Journal: Science Advances

Article Title: Neuronal Reg3β/macrophage TNF-α–mediated positive feedback signaling contributes to pain chronicity in a rat model of CRPS-I

doi: 10.1126/sciadv.adu4270

Figure Lengend Snippet: ( A ) Experiments schedule. ( B ) p-STAT3 and STAT3 expression in ipsilateral L4-L6 DRG of sham and CPIP model rats. n = 5 rats per group. ( C ) p-STAT3 immunostaining (green) in DRG of sham + Veh, CPIP + Veh, and CPIP + ETA groups. Neurons were stained with Nissl (red). ( D ) Normalized fluorescence intensity of p-STAT3 immunostaining in DRG of three groups as in (C). n = 5 rats per group. ( E ) p-STAT3 and STAT3 expression in ipsilateral L4-L6 DRG of three groups. n = 6 rats per group. ( F ) Overlapping of p-STAT3 + cells (green) with p-Syk + cells (red) and neurons (by Nissl, purple) in DRG of CPIP model rats. White arrows indicate overlapped cells. ( G ) Reg3β immunostaining (in red) in DRG of sham + Veh, CPIP + Veh, and CPIP + S3I-201 groups. ( H ) Normalized fluorescence intensity of Reg3β in DRG of three groups as in (G). n = 6 to 7 rats per group. ( I ) Immunostaining showing overlapping of p-STAT3 + cells (green) with Reg3β + cells (red) in DRG of CPIP model rats. Purple: DAPI. White arrows indicate overlapped cells. ( J ) CD68 immunostaining (red) in DRG of sham + Veh, CPIP + Veh, and CPIP + S3I-201 groups. DAPI in purple. ( K ) The number of CD68 + cells in DRG of three groups as in (J). n = 6 rats per group. * P < 0.05 and ** P < 0.01. ( L ) 50% PWT changes in three groups. n = 6 rats per group.** P < 0.01 versus sham + Veh group. ## P < 0.01 versus CPIP + Veh group. Scale bar is as indicated. One-way ANOVA followed by Tukey’s post hoc test for [(D), (E), (H), and (K)]. Two-way ANOVA followed by Tukey’s post hoc test for (L). Student’s unpaired t test for (B).

Article Snippet: Cells were incubated with CD68 Ab (1:500; RRID: AB_3170485, #NB100-683PE, NOVUS Biologicals, USA) 4°C for 1 hour.

Techniques: Expressing, Immunostaining, Staining, Fluorescence

Journal: ACS Chemical Neuroscience

Article Title: Kisspeptin Prevention of Amyloid-? Peptide Neurotoxicity in Vitro

doi: 10.1021/cn300045d

Figure Lengend Snippet: Binding of KP to Aβ, PrP and IAPP peptides. Alignment of the human metastasis-suppressor KiSS-1 preproprotein sequence (NP_002247.3) with the human catalase sequence (NP_001743.1) is shown in A. The red box highlights the region of KP that prevents Aβ, PrP, and IAPP toxicity; blue and green boxes highlights the Gly-Ala-Ile-Ile region that binds catalase in schemes A and B respectively; the black box highlights Aβ 31–35, which inhibits Aβ 1–42 binding to catalase. Immunoplates were coated with Aβ 1–40, Aβ 1–28, Aβ 29–40, Aβ 25–35, PrP 106–126, PrP 118–135, IAPP 1–37, IAPP 20–29, A-Bri 1–34, or A-Dan 1–34 fibrils. Coated plates were incubated with either biotinylated KP 45–54 (B) alone (open columns) or in the presence of unlabeled KP 45–54 (closed blue columns) and bound material determined by EIA. Plates coated with KP 1–54, KP 27–54, KP 42–54, KP 45–54, KP 45–50, KP 45–47, KP 47–50, or NPFF (C) were incubated with biotinylated Aβ 1–42 (open columns), biotinylated PrP 106–126 (red columns), or biotinylated IAPP 1–37 (black columns) and bound material determined by EIA. All results are expressed as the mean ± SEM (n = 8). (* = P < 0.05 vs control (buffer alone); † = P < 0.05 vs biotinylated KP 45–54; one-way ANOVA.)

Article Snippet: Test Peptides N-Terminally biotinylated KP 1–54, KP 45–54, Aβ 1–42, Aβ 1–40, PrP 106–126, and IAPP 1–37 were purchased from Bachem or Alpha Diagnostics.

Techniques: Binding Assay, Sequencing, Incubation, Control

Journal: ACS Chemical Neuroscience

Article Title: Kisspeptin Prevention of Amyloid-? Peptide Neurotoxicity in Vitro

doi: 10.1021/cn300045d

Figure Lengend Snippet: Effects of KP on Congo red binding to Aβ, PrP, and IAPP. The Aβ 1–40, Aβ 1–28, Aβ 25–35, Aβ 29–40, PrP 106–126, PrP 118–135, IAPP 1–37, IAPP 20–29, A-Bri 1–34, and A-Dan 1–34 peptides were dissolved in PBS and incubated at 37 °C for 24 h, with constant oscillation, with or without KP 45–50. Congo red binding assays (A) were performed with amyloid peptides alone (open columns) or in the presence of KP 45–50 (closed blue columns). The effect of Congo red (0–100 mM) on biotinylated Aβ 1–42 binding to KP 45–54 (B) was determined by EIA. All results are expressed as the mean ± SEM (n = 8). (* = P < 0.05 vs amyloid fibrils alone; one-way ANOVA.)

Article Snippet: Test Peptides N-Terminally biotinylated KP 1–54, KP 45–54, Aβ 1–42, Aβ 1–40, PrP 106–126, and IAPP 1–37 were purchased from Bachem or Alpha Diagnostics.

Techniques: Binding Assay, Incubation

Journal: Journal of Biomedical Science

Article Title: Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35

doi: 10.1186/s12929-023-00910-w

Figure Lengend Snippet: The top 10 genes with differentially expressed levels in wild-type, tigecycline-resistant strains (109 and 200) and tigecycline-resistant mutants (109-IR and 200-IR)

Article Snippet: KP1_1491 , MBL fold metallo-hydrolase , 244.571 , 14.271 , 2441.961 , 131.245 , 17.950 , 2404.509.

Techniques:

Journal: ACS Chemical Neuroscience

Article Title: Kisspeptin Prevention of Amyloid-? Peptide Neurotoxicity in Vitro

doi: 10.1021/cn300045d

Figure Lengend Snippet: Binding of KP to Aβ, PrP and IAPP peptides. Alignment of the human metastasis-suppressor KiSS-1 preproprotein sequence (NP_002247.3) with the human catalase sequence (NP_001743.1) is shown in A. The red box highlights the region of KP that prevents Aβ, PrP, and IAPP toxicity; blue and green boxes highlights the Gly-Ala-Ile-Ile region that binds catalase in schemes A and B respectively; the black box highlights Aβ 31–35, which inhibits Aβ 1–42 binding to catalase. Immunoplates were coated with Aβ 1–40, Aβ 1–28, Aβ 29–40, Aβ 25–35, PrP 106–126, PrP 118–135, IAPP 1–37, IAPP 20–29, A-Bri 1–34, or A-Dan 1–34 fibrils. Coated plates were incubated with either biotinylated KP 45–54 (B) alone (open columns) or in the presence of unlabeled KP 45–54 (closed blue columns) and bound material determined by EIA. Plates coated with KP 1–54, KP 27–54, KP 42–54, KP 45–54, KP 45–50, KP 45–47, KP 47–50, or NPFF (C) were incubated with biotinylated Aβ 1–42 (open columns), biotinylated PrP 106–126 (red columns), or biotinylated IAPP 1–37 (black columns) and bound material determined by EIA. All results are expressed as the mean ± SEM (n = 8). (* = P < 0.05 vs control (buffer alone); † = P < 0.05 vs biotinylated KP 45–54; one-way ANOVA.)

Article Snippet: N-Terminally biotinylated KP 1–54, KP 45–54, Aβ 1–42, Aβ 1–40, PrP 106–126, and IAPP 1–37 were purchased from Bachem or Alpha Diagnostics.

Techniques: Binding Assay, Sequencing, Incubation, Control

Journal: ACS Chemical Neuroscience

Article Title: Kisspeptin Prevention of Amyloid-? Peptide Neurotoxicity in Vitro

doi: 10.1021/cn300045d

Figure Lengend Snippet: Effects of KP on Congo red binding to Aβ, PrP, and IAPP. The Aβ 1–40, Aβ 1–28, Aβ 25–35, Aβ 29–40, PrP 106–126, PrP 118–135, IAPP 1–37, IAPP 20–29, A-Bri 1–34, and A-Dan 1–34 peptides were dissolved in PBS and incubated at 37 °C for 24 h, with constant oscillation, with or without KP 45–50. Congo red binding assays (A) were performed with amyloid peptides alone (open columns) or in the presence of KP 45–50 (closed blue columns). The effect of Congo red (0–100 mM) on biotinylated Aβ 1–42 binding to KP 45–54 (B) was determined by EIA. All results are expressed as the mean ± SEM (n = 8). (* = P < 0.05 vs amyloid fibrils alone; one-way ANOVA.)

Article Snippet: N-Terminally biotinylated KP 1–54, KP 45–54, Aβ 1–42, Aβ 1–40, PrP 106–126, and IAPP 1–37 were purchased from Bachem or Alpha Diagnostics.

Techniques: Binding Assay, Incubation