Journal: Frontiers in Pharmacology

Article Title: Jianpi Yangzheng Xiaozheng decoction alleviates gastric cancer progression via suppressing exosomal PD-L1

doi: 10.3389/fphar.2023.1159829

Figure Lengend Snippet: JPYZXZ reduced the number of MDSCs in tumor bearing mice. (A,B) Percentage of MDSCs (CD45 + CD11b+Gr1+) in blood determined using flow cytometry. (C,D) Percentage of MDSCs (CD45 + CD11b+Gr1+) in tumors determined using flow cytometry. (E) Immunofluorescence staining for DAPI (nucleus), CD11b, Gr-1, and PD-L1 in tumor tissue sections from the xenograft tumor model. Data are represented as the mean ± SEM, n = 5 mice or n = 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The single-cell suspensions (1 × 10 7 cells/mL) were stained with fluorochrome-coupled antibodies AF488-CD45 (Elabscience, E-AB-F1136L), PE/Dazzle™594-CD11b (BioLegend, 101256), and APC-Gr-1 (BioLegend, 108412) for 30 min at 4°C.

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC-Vio770 Recombinant human anti B220(clone REA755) , Miltenyi Biotec , Cat# 130–110-849; RRID:AB_2658286.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: Aging limits T RM establishment within the brain. 8 week. adult or 18 months aged C57BL/6 mice were inoculated with 10 3 pfu MHV‐A59 i.n., CD8 + T cells harvested from the spleen 7 DPI and then adoptively transferred intravenously (i.v.) via the tail vein into either 8 week. or 18 months old animals that were infected with (a,b) 10 4 pfu or (c,d) 10 3 pfu MHV‐A59 1 day prior. (a, c) Survival and (b, d) weight change were monitored for 30 DPI. (e) Representative flow cytometry plots of lymphocytes stained for CD8, then CD45.1 and CD45.2 to distinguish between host and donor cells isolated from the brains of 8 week. or 18 months old animals at 30 DPI following infection with 10 3 pfu MHV‐A59 and adoptive transfer of 8 week. or 18 months cells as indicated. (f) Frequency and (g) total number of total CD8 + cells and total transferred cells. (h) Representative flow cytometry histograms of CD103 expression by CD8 + T cells present in the brain of 8 week. or 18 months animals 30 DPI following infection and adoptive transfer as described. (i) Quantification of CD103 MFI by host or donor cells within the same host. Data are representative of 1 independent experiment with each data point representing an individual animal. Survival assessed by Log‐rank Mantel‐Cox assessment, weight change and flow cytometry assessment conducted according to two‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, Adoptive Transfer Assay, Expressing

Journal: Aging Cell

Article Title: Compromised CD8+ T cell immunity in the aged brain increases severity of neurotropic coronavirus infection and postinfectious cognitive impairment

doi: 10.1111/acel.14409

Figure Lengend Snippet: CD8 + T cells mediate neuronal apoptosis following MHV‐A59 infection. (a) Representative IHC of DAPI, NeuN, and CD8a in the DG and cortex of 8 week or 18 months old animals at (a) 12 DPI and (b) 30 DPI. (c) Representative flow cytometry plots of CD45 + CD8 + cells stained for IFN‐γ isolated from the brains of 8 week or 18 months old, mock or MHV‐A59 infected animals at 30 DPI following 4 h. PMA/ionomycin stimulation. (d) Quantification of percent CD8 + CD44 + cells positive for IFN‐γ. (e) Representative immunocytochemical (ICC) staining for NeuN and TUNEL of primary cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without coculture with CD8 + T cells purified from the spleen of an 8 week old animal at 7 DPI with 10 3 pfu MHV‐A59. Quantification of ICC images: (f) total number NeuN + neuronal nuclei, (g) total number TUNEL + NeuN + neuronal nuclei, (h) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. (i) Representative ICC of NeuN and TUNEL staining of primary embryonic cortical neurons following no treatment or infection with MHV‐A59 MOI 0.5, with or without naïve or PMA/ionomycin stimulated CD8 + T cells purified from the spleen of an 8 week old uninfected animal. Quantification of ICC images: (j) total number NeuN + neuronal nuclei, (k) total number TUNEL + NeuN + neuronal nuclei, and (l) proportion of TUNEL + NeuN + neuronal nuclei of total NeuN + neuronal nuclei present. Data are representative of three independent experiments with each data point representing an individual sample. All images were taken at 40X magnification and scale bar = 100 μm. Three images were captured per sample and averaged. NeuN + and TUNEL + NeuN + neuronal nuclei quantified in ImageJ software using the Cell Counter plugin. Statistics according to unpaired one‐way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Tetramer staining was carried out at room temperature for 20 min in conjugation with other surface staining antibodies: CD8a (53–6.7, APC‐Cy7), CD4 (RM4‐5, APC), CD44 (IM7, PE‐Cy7), CD45 (30‐F11, APC), CD45.1 (20–0453, APC), CD45.2 (60–0454, PE‐Cy‐7), CD11b (M1/70, FITC), P2RY12 (S16007D, PE), CD68 (FA‐11, BV‐421), MHC II (M5/114.15.2, PerCP‐Cy5.5), obtained from Cytek Biosciences or eBioscience, then washed with 1× PBS and fixed with 2% paraformaldehyde (PFA).

Techniques: Infection, Flow Cytometry, Staining, Isolation, TUNEL Assay, Purification, Software

Journal: FEBS Open Bio

Article Title: Neutrophil and macrophage crosstalk might be a potential target for liver regeneration

doi: 10.1002/2211-5463.13803

Figure Lengend Snippet: Neutrophils increased after PH. (A) Flow cytometry showed the percentage of neutrophils (Ly6G + CD11b + ) among total immune cells (CD45 + ) after PH ( n = 6, * P < 0.05). (B) Immunofluorescence staining of Ly6G + neutrophils time after PH ( n = 3, * P < 0.05). (C) Schematic representation of neutrophil depletion by PH (control: rat IgG2a isotype; anti‐Gr1: anti‐Gri antibody; combo: anti‐Ly6G antibody with anti‐rat IgG2a). (D) Flow cytometry showed the percentage of neutrophils (Ly6G + CD11b + ) in total immune cells in different groups (CD45 + ) ( n = 6, * P < 0.05). (E) Immunofluorescence staining of Ly6G + neutrophils in different groups ( n = 3, * P < 0.05).

Article Snippet: Primary antibodies against CD45 (dilution 1 : 100; 75‐0451‐U100; TONBO, Tokyo, Japan), CD11b (dilution 1 : 100; 85‐0112‐U100; TONBO), Ly6G (dilution 1 : 100; 127633; BioLegend, San Diego, CA, USA), F4/80 (dilution 1 : 100; 123137; BioLegend), LY6C (dilution 1 : 100; 20‐5932‐U100; TONBO) and CLEC4F (dilution 1 : 100; 156804; BioLegend) were carried out in FACS buffer (4 °C for 60 min).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Control

Journal: FEBS Open Bio

Article Title: Neutrophil and macrophage crosstalk might be a potential target for liver regeneration

doi: 10.1002/2211-5463.13803

Figure Lengend Snippet: The interaction between neutrophils and macrophages enhances liver regeneration. (A) Network diagrams showing cell–cell interactions indicated by arrows (edges) pointing in the source‐to‐target direction. The thickness indicates the sum of the weighted paths between populations, and the color of the arrows corresponds to the source. Network diagrams for clusters 10–18 are shown. (B) Flow cytometry was used to determine the percentage of macrophages (F4/80 + CD11b + ) in total immune cells (CD45 + ) in the different groups ( n = 6, * P < 0.05). (C) Flow cytometry revealed the percentage of Kupffer cells (CLEC4F + ) in macrophages in the different groups (F4/80 + CD11b + CD45 + ) ( n = 6, * P < 0.05). (D) Flow cytometry revealed the percentage of monocytes (Ly6C + CD11b + ) among the total immune cells in the different groups (CD45+) ( n = 6, * P < 0.05). (E) The mRNA expression levels of Ccl3, Ccl5 and Cxcl1 were examined using qPCR analysis (the genes were normalized to GAPDH mRNA levels in each sample. Ccl3, C‐C motif chemokine ligand 3; Ccl5, C‐C motif chemokine ligand 5; Cxcl1, C‐X‐C motif chemokine ligand 1; n = 3, * P < 0.05). (F) Macrophages cultured without neutrophils and co‐culture of neutrophils and macrophages ( n = 3, * P < 0.05).

Article Snippet: Primary antibodies against CD45 (dilution 1 : 100; 75‐0451‐U100; TONBO, Tokyo, Japan), CD11b (dilution 1 : 100; 85‐0112‐U100; TONBO), Ly6G (dilution 1 : 100; 127633; BioLegend, San Diego, CA, USA), F4/80 (dilution 1 : 100; 123137; BioLegend), LY6C (dilution 1 : 100; 20‐5932‐U100; TONBO) and CLEC4F (dilution 1 : 100; 156804; BioLegend) were carried out in FACS buffer (4 °C for 60 min).

Techniques: Flow Cytometry, Expressing, Cell Culture, Co-Culture Assay