ic21 murine peritoneal macrophage cell line  (ATCC)

Journal: Journal for Immunotherapy of Cancer

Article Title: Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer

doi: 10.1136/jitc-2022-006170

Figure Lengend Snippet: Pten -deficient ovarian cancer cells alter their tumor immune microenvironment towards a suppressive state via polarizing macrophages to M2-like phenotype. Total RNA from untreated tumors of different genotypes were subjected to NanoString gene expression profiling using the PanCancer immune gene panel displayed as a (A) volcano plot showing differential expression pattern of genes associated with macrophage function. (B) Proportion of cells derived from ascites of mice injected with ID8- Trp53 −/− ; Pten −/− and ID8- Trp53 −/− ; Brca1 −/− cells expressing CD45+CD11b+F4/80+cells. (C) MCP-1 chemokine levels derived from the ascites fluid of untreated mice injected with ID8- Trp53 −/− ; Pten −/− and ID8- Trp53 −/− ; Brca1 −/− cells. (D) Transwell migration assay of IC21 peritoneal macrophage cells stimulated (from left to right) with media control (Dulbecco’s Modified Eagle’s Medium), positive control (20 ng/mL MCP-1), or 72 hours conditioned media of ID8- Trp53 −/− ; Pten −/− and ID8- Trp53 −/− ; Brca1 −/− cells. Stimulated for 16 hours; images 20x magnification. Flow cytometric analysis of IC21 macrophage proportions expressing (E) CD45+F4/80+CD206+ post-stimulation with concentrated ascites derived from mice injected with different genotypes of HGSC cells. US, unstimulated IC21 cells; M1 positive control represented by IC21 cells stimulated with IFN-ɣ (50 ng/mL) and LPS (100 ng/mL) in complete media; M2 positive control represented by IC21 cells stimulated with IL-4 (10 ng/mL) and IL-10 (20 ng/mL) in complete media. Averages of triplicate wells/experiment for three repeated experiments displayed. Mann-Whitney non-parametric test was used for (B–C) *p<0.05 **p<0.005, ***p<0.001, ****p<0.0001. One-way analysis of variance applied for D and E. Mean±SD. HGSC, high-grade serous ovarian cancer; IFN, interferon; IL, interleukin; MCP-1, macrophage chemoattractant protein 1; ns, not significant; PTEN, phosphatase and tensin homolog.

Article Snippet: IC21 murine peritoneal macrophage cell line was obtained from American Type Culture Collection (Manassas, Virginia, USA) and cultured in RPMI 1640 (Sigma-Aldrich, Ontario, Canada) supplemented with 10% FBS and 1% penicillin/streptomycin.

Techniques: Gene Expression, Quantitative Proteomics, Derivative Assay, Injection, Expressing, Transwell Migration Assay, Control, Modification, Positive Control, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: IL-36γ is secreted through an unconventional pathway using the Gasdermin D and P2X7R membrane pores

doi: 10.3389/fimmu.2022.979749

Figure Lengend Snippet: IL-36γ is secreted in response to ATP in a non-conventional pathway. (A) IL-36γ protein expression in lysates (L) and supernatants (S) of LPS or LPS/ATP activated macrophages. Densitometry of IL-36γ intensity in lysates (B) and supernatants (C) of cell stimulated with LPS or LPS/ATP (L/A). (D) IF of IC21 macrophages stimulated with LPS/ATP (L/A) and treated with monensin (Mon) or brefeldin A (BfA). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Murine macrophages cell line IC21 (TIB-186) was cultured according to the manufacturer’s instructions (ATCC, VA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: IL-36γ is secreted through an unconventional pathway using the Gasdermin D and P2X7R membrane pores

doi: 10.3389/fimmu.2022.979749

Figure Lengend Snippet: IL-36γ is localized in the cytoplasm of IC21 macrophages. IL-36γ localization in IC21 macrophages in control, LPS, LPS/ATP (L/A), LPS/ATP+A438, and LPS/ATP+NSA conditions. nuclei staining (DAPI), IL-36γ (Alexa Fluor594).

Article Snippet: Murine macrophages cell line IC21 (TIB-186) was cultured according to the manufacturer’s instructions (ATCC, VA).

Techniques: Control, Staining

Journal: Advanced healthcare materials

Article Title: TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease

doi: 10.1002/adhm.201801271

Figure Lengend Snippet: Characterization of EVs isolated from TPP1-transfected macrophages (EV-TPP1-t). TPP1-transfected macrophages and their EVs display elevated levels of A) TPP1 protein and B) enzymatic activity between second and seventh day post-transfection. IC21 macrophages were transfected with TPP1-encoding pDNA (2 μg mL−1 pDNA with GenePorter 3K for 4 h), and (A) TPP1 protein expression and (B) TPP1 enzymatic activity were assessed in the parental cells (white bars) and EVs (black bars) using (A) ELISA and (B) a TPP1 substrate, AF-AMC (400 μM), respectively. (A) TPP1 protein or (B) activity levels in nontransfected cells (dashed line) or EVs secreted by them (solid line) were also recorded. For EVs’ activity, the levels are normalized to the number of cells used to isolate these EVs. C) Increased stability of TPP1 in EVs (solid line) compared to EV-free TPP1 (dashed line) upon incubation with pronase protease from Streptomyces Greseus (4 × 10−5 M). D) Round morphology of EVs released by empty-transfected macrophages and TPP1-transfected macrophages. E) Quantitative PCR analysis indicated a significant amount of TPP1-encoding pDNA incorporated in EVs from pretransfected macrophages. (A–C) Statistical significance *p < 0.05, or **p < 0.005 compared to TPP1 levels in nontransfected cells or (A,B) EVs, or (E) EV-free TPP1.

Article Snippet: Cells: IC21 cell line derived by transformation of normal C57BL/6 mouse peritoneal macrophages with SV40, and neuronal PC12 rat adrenal pheochromocytoma cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, South Logan, UT, USA) supplemented with 10% FBS, and 1% (v/v) of both penicillin and streptomycin.

Techniques: Isolation, Transfection, Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Real-time Polymerase Chain Reaction

Journal: Advanced healthcare materials

Article Title: TPP1 Delivery to Lysosomes with Extracellular Vesicles and their Enhanced Brain Distribution in the Animal Model of Batten Disease

doi: 10.1002/adhm.201801271

Figure Lengend Snippet: Characterization of EV-TPP1-l obtained by TPP1 loading into naïve EVs by either sonication or saponin permeabilization: A) TPP1 enzymatic activity and NTA parameters, B) morphology by AFM, C) TPP1 release, and D) TPP1 stability in the presence of pronase protease from Streptomyces Creseus (4 × 10−5 M) for sonicated EV-TPP1 versus free TPP1. (A–D) IC21 macrophage-derived EVs (1011 particles mL−1) were loaded with TPP1 (20 μg/100 μL) by sonication in water bath, or saponin permeabilization (0.4 mg mL−1). (B) The bar: 200 nm. (D) TPP1 activity was measured using AF-AMC (400 × 10−6 M) as a substrate. Statistical comparisons of (A) loaded EV-TPP1 versus naive EVs or (D) treated versus untreated with pronase TPP1 formulations: *p < 0.05, **p < 0.005 (n = 4).

Article Snippet: Cells: IC21 cell line derived by transformation of normal C57BL/6 mouse peritoneal macrophages with SV40, and neuronal PC12 rat adrenal pheochromocytoma cell line were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, South Logan, UT, USA) supplemented with 10% FBS, and 1% (v/v) of both penicillin and streptomycin.

Techniques: Sonication, Activity Assay, Derivative Assay