Journal: Nature immunology

Article Title: Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly

doi: 10.1038/s41590-020-0646-0

Figure Lengend Snippet: List of antibodies used in flow cytometry.

Article Snippet: Stains were performed for 30 minutes at 4 o C and followed by two washes with PBS containing 2% FCS and 2 mM EDTA before fixation in 0.5% PFA in PBS. table ft1 table-wrap mode="anchored" t5 Table 2.2: caption a7 Name Supplier Type Clone CD3 Biolegend Mouse IgG2a HIT3a CD11b Biolegend Mouse IgG1 ICRF44 CD14 Biolegend Mouse IgG2a M5E2 CD14 Biolegend Mouse IgG1 HCD14 CD16 Biolegend Mouse IgG1 3G8 CD19 Biolegend Mouse IgG1 HIB19 CD36 BD Biosciences Mouse IgM CB38 (NL07) CD51 (ITGAV) Miltenyi Biotec Recombinant IgG1 REA181 CD56 Biolegend Mouse IgG2a MEM-188 CD62L Biolegend Mouse IgG1 DREG-56 CD66b Biolegend Mouse IgM G10F5 CD95 (Fas) Biolegend Mouse IgG1 DX2 CD120a (TNFRI) Miltenyi Biotec Recombinant IgG1 REA252 CD120b (TNFRII) Biolegend Rat IgG2a 3G7A02 CD163 Biolegend Mouse IgG1 RM3/1 CD181 (CXCR1) Biolegend Mouse IgG2b 8F1/CXCR1 CD182 (CXCR2) Biolegend Mouse IgG1 5E8/CXCR2 CD192 (CCR2) BD Biosciences Mouse IgG2a LS132.1D9 (1D9) CD197 (CCR7) Biolegend Mouse IgG2a G043H7 CD206 Biolegend Mouse IgG1 15-2 Siglec-8 Biolegend Mouse IgG1 837535 HLA-DR BD Biosciences Mouse IgG2a G46-6 MerTK eBioscience Rat IgG2a DS5MMER TIM-4 Biolegend Mouse IgG1 9F4 P-p38 eBioscience Mouse IgG2b 4NIT4KK p65 (RelA) Biolegend Mouse IgG2b 14G10A21 P-STAT-3 (Tyr705) Biolegend Mouse IgG1 13A3-1 Live/Dead Zombie NIR Biolegend N/A N/A Open in a separate window List of antibodies used in flow cytometry.

Techniques: Cytometry, Recombinant

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Human Milk-derived Peptide Drives Rapid Regulation of Macrophage Inflammation Responses in the Neonatal Intestine

doi: 10.1016/j.jcmgh.2024.101420

Figure Lengend Snippet: CASB 135-150 mitigates the pro-inflammatory response in intestinal macrophages. ( A ) The neonatal rats were gavaged with either saline or saline supplementation with human milk EVs. After a 2-hour period, the ileums were collected for immunofluorescence analysis of IBA1, β-casein, and DAPI. n = 3. ( B–C ) Intestinal lamina propria lymphocytes were isolated from NEC animals that received CASB 135-150 or CASB 135-150 -Mut treatment (20 mg/kg/day for 4 days). CD45+F4/80+CD11b/c+ macrophages were gated for analysis of TNF-α by flow cytometry ( B ) and the percentage of TNF-α+ cells quantification, n = 3 ( C ). ( D–F ) BMDMs were pre-treated with CASB 135-150 at the indicated concentrations for 1 hour, followed by stimulation with LPS (10 ng/ml) for 6 hours. Subsequently, the levels of inflammation factors were assessed using qRT-PCR, GAPDH as a reference gene, n = 3. ( G–H ) BMDMs were pre-treated with CASB 135-150 at a concentration of 50 μM, followed by LPS (10 ng/ml) treatment for 24 hours. Afterward, the cells were collected for flow cytometry analysis of CD86 and CD206 ( G ) and quantification ( H ). n = 3 Data are presented as mean ± SD. Statistical significance was determined using 1-way ANOVA with Tukey’s post hoc test. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: We then centrifuged and discarded the supernatant, added 100 μL of fixation and permeabilization solution (554714, BD), resuspended the cells, incubated at 4 °C in the dark for 40 minutes, centrifuged and discarded the supernatant, added 150 μL of permeabilization wash buffer (554714, BD), centrifuged and discarded the supernatant, then added the diluted Alexa Fluor 700 anti-TNF-α antibody (NBP1-19532AF700, Novus Biologicals, 1:100 dilution), resuspended the cells, and incubated in the dark for 60 minutes.

Techniques: Saline, Immunofluorescence, Isolation, Flow Cytometry, Quantitative RT-PCR, Concentration Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Human Milk-derived Peptide Drives Rapid Regulation of Macrophage Inflammation Responses in the Neonatal Intestine

doi: 10.1016/j.jcmgh.2024.101420

Figure Lengend Snippet: FHL2 overexpression amplifies the anti-inflammatory effects controlled by CASB 135-150 treatment. ( A–B ) RAW264.7 cells were overexpressed with FHL2 for 48 hours, followed by pre-treatment with CASB 135-150 for 1 hour and subsequent exposure to LPS for 6 hours. The production of Tnf-α ( A ) and IL-6 ( B ) was assessed using qPCR. GAPDH as a reference gene. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001.

Article Snippet: We then centrifuged and discarded the supernatant, added 100 μL of fixation and permeabilization solution (554714, BD), resuspended the cells, incubated at 4 °C in the dark for 40 minutes, centrifuged and discarded the supernatant, added 150 μL of permeabilization wash buffer (554714, BD), centrifuged and discarded the supernatant, then added the diluted Alexa Fluor 700 anti-TNF-α antibody (NBP1-19532AF700, Novus Biologicals, 1:100 dilution), resuspended the cells, and incubated in the dark for 60 minutes.

Techniques: Over Expression

Journal: Immunity

Article Title: The Fc domain of immunoglobulin is sufficient to bridge NK cells with virally infected cells

doi: 10.1016/j.immuni.2017.06.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Detailed methods are provided in the online version of this paper and include the following: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BV421 anti-human CD3 (clone HIT3a) BD Cat# 740073 APC-H7 anti-human CD14 (M5E2) BD Cat# 561384 PE anti-human CD19 (clone HIB19) BD Cat# 555413 PC7 anti-human CD56 (clone N901) Beckman Coulter Cat# {"type":"entrez-nucleotide","attrs":{"text":"A51078","term_id":"2303855","term_text":"A51078"}} A51078 FITC anti-human CD69 (clone FN50) BD Cat# 555530 PE anti-human CD62L (clone DREG-56) BD Cat# 555544 V421anti-human CD107a (clone H4a3) Biolegend Cat# 328626 APC/Cy7 anti-human CD16 (clone 3G8) Biolegend Cat# 302018 FITC CD64 (clone 10.1) BD Cat# 555527 PE Ms IgG1, κ (clone MOPC-21) BD Cat# 555749 APC anti-human IgG (polyclonal) Jackson ImmunoResearch Cat# 209605098 APC anti-human IFNgamma (clone B27) BD Cat# 554702 Anti-HSV1 gE (clone 9H3) abcam Cat# Ab6510 FITC anti-human CD3ζ (clone 6B10.2) Biolegend Cat# 644104 APC anti-human CD3zeta (pY142) (clone K25-407.69) BD Cat# 558489 Anti asialo gm1 Biolegend Cat# 146002 PE anti-human CD120a (TNF-RI) (clone REA252) Miltenyi Biotech Cat# 130-106-360 IgG1, Kappa from human myeloma plasma Sigma Cat# I5154-1MG IgG3 Kappa Sigma Cat# I5654 Immune globulin, GamaStan S/D GRIFOLS N/A Rituximab Genentech N/A Obinutuzumab Janssen N/A Immunoglobulin G-Fc Fragment; Purified Scripps Laboratories Cat# I2724 Bacterial and Virus Strains HSV1 F strain ATCC Cat# VR-733 HSV1 Us8- F strain Suenaga et al., 2014 N/A R8411 (HSV1 F strain expresses luciferase) Zerboni et al., 2013 N/A MCMV Orr MT et al., 2010 N/A Staphylococcus newman strain (wt) ATCC Cat# 25904 Staphylococcus newman strain (spa-) Patel et al., 1987 N/A Biological Samples Umbilical cord blood American Red Cross, Columbus, OH, USA N/A Chemicals, Peptides, and Recombinant Proteins FCGR3A (CD16a), 176 Phe/Val Recombinant Human Protein, His.

Techniques: Clinical Proteomics, Purification, Virus, Luciferase, Recombinant, Cell Isolation, Enzyme-linked Immunosorbent Assay, Gel Extraction, Magnetic Beads, Plasmid Preparation, Software

Journal: Journal of Neuroinflammation

Article Title: Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat

doi: 10.1186/s12974-017-0822-9

Figure Lengend Snippet: List of primary and secondary antibodies used for Western blot analysis

Article Snippet: TNF-α , rabbit , Sino Biological , 80045-RP02 , 1:500 , Overnight 4 °C.

Techniques: Western Blot, Incubation

Journal: Journal of Neuroinflammation

Article Title: Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat

doi: 10.1186/s12974-017-0822-9

Figure Lengend Snippet: Suppressed activation of glial cells and TNF-α production by inhibition of MyD88 in rat SDH after CCI. a Immunostaining showing inhibitory effects of MIP on activation of microglial cells (IBA1), and astrocytes (GFAP). Scale bar: 20 μm. b Western blot showing inhibitory effects of MIP on CCI-induced increased protein level of IBA1, GFAP, and TNF-α. c Data summary of B. Others are the same as Fig.

Article Snippet: TNF-α , rabbit , Sino Biological , 80045-RP02 , 1:500 , Overnight 4 °C.

Techniques: Activation Assay, Inhibition, Immunostaining, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat

doi: 10.1186/s12974-017-0822-9

Figure Lengend Snippet: Schematic illustration demonstrates MyD88-dependent signaling pathways of neuropathic pain induced by CCI. Nerve injury produces abundant HMGB1and IL-1β in the DRG and SDH. The binding of HMGB1 and IL-1β to their receptors (TLR2/4 andIL-1R, respectively) activates MyD88 in the DRG and SDH, which phosphorylate NF-κB p65 and ERK. Phosphorylated NF-κB p65 subsequently enter the nucleus to regulate the expression of proinflammation cytokines such as TNF-α. Phosphorylated ERK enter the nucleus to induce transcription factors such as AP-1, which regulates the expression of certain cytokines and activates glial cells. All these signaling events consequently result in central and peripheral sensitizations that produce neuropathic pain

Article Snippet: TNF-α , rabbit , Sino Biological , 80045-RP02 , 1:500 , Overnight 4 °C.

Techniques: Binding Assay, Expressing