Journal: Nature Communications

Article Title: p140Cap inhibits β-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response

doi: 10.1038/s41467-023-37824-y

Figure Lengend Snippet: a Effect of p140Cap over-expression on tumor growth and metastasis in TuBo and 4T1 BC cell models. TuBo (10 5 ) or 4T1 (10 4 ) mock and p140Cap cells were inoculated into the fat pad of 8-weeks-old female BALB/c mice. Tumor growth was monitored and tumor size was measured every two days from tumor onset (TuBo, n = 11; 4T1, n = 5; data are represented for n = x mices, two-tailed unpaired t test). For metastasis analysis in TuBo mock and p140Cap tumor-bearing mice, tumors were surgically removed when they reached 10 mm diameter and mice were kept alive. After 5 weeks, mice were sacrified and lungs were explanted and analyzed. For metastasis analysis of 4T1 mock and p140Cap tumors-bearing mice, lungs were analyzed 22 and 30 days post-injection for mock and p140Cap cells, respectively. Representative dot plots show the number of lung metastasis as mean ± SEM (Standard Error of the Mean; TuBo, n = 5; 4T1, n = 10; 2way ANOVA). b , c Flow cytometry analysis for M1- and M2-macrophages, CD4 + and CD8 + T-Lymphocytes, Natural Killer cells in tumor-bearing mice. Representative dot plots show the percentage (%) of tumor infiltrated M1- and M2-macrophages, CD4 + and CD8 + T-Lymphocytes, Natural Killer (NK) cells, normalized on CD45 + cells in TuBo mock and p140Cap tumor-bearing mice in panel ( b ) ( n = 8/M1, CD4 + and CD8 + and n = 9/NK, n = 5/M2) and 4T1 mock and p140Cap tumor-bearing mice in panel ( c ) ( n = 5/group). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test). For TuBo mock and p140Cap tumors, the analysis was performed at day 26 or 32, respectively, while for 4T1 mock and p140Cap tumors at day 12. d Flow cytometry analysis for PMN-MDSCs (CD11b + Ly6G + Ly6C low ) and for M-MDSCs (CD11b + Ly6G − Ly6C + ) normalized on CD45 + cells, in tumor-bearing mice. Representative dot plots show the percentage of tumor infiltrated PMN-MDSCs and M-MDSCs cells in TuBo and 4T1 mock and p140Cap tumor-bearing mice, as described in panels ( b , c ) (TuBo n = 8/group; 4T1 n = 7/PMN-MDSCs and n = 8/M-MDSCs). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test.

Article Snippet: The following antibodies were used (at a dilution of 1:200): CD45-VioGreen (Cat#130-110-803), CD11b-FITC (Cat#130-110-803), Ly6G-VioBlue (Cat#130-119-986), Ly6C-APC-Vio770 (Cat#130-121-439), F4/80-PE-Vio770 (Cat#130-118-320), MHC-II-APC (Cat#130-102-139), CD3-FITC (Cat#130-119-135), CD4-APC-Vio770 (Cat#130-119-134), CD8-VioBlue (Cat#130-123-865), CD49b-PE (Cat#130-123-702) all from Miltenyi Biotec (Miltenyi Biotec B.V. & Co. Bologna, Italy), while CD206-PE (Cat#141706) was from BioLegend (BioLegend, San Diego CA, USA).

Techniques: Over Expression, Two Tailed Test, Injection, Flow Cytometry

Journal: Nature Communications

Article Title: p140Cap inhibits β-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response

doi: 10.1038/s41467-023-37824-y

Figure Lengend Snippet: a , b Flow cytometry analysis for PMN-MDSCs (CD11b + Ly6G + Ly6C low ), normalized on CD45 + cells, in lungs, blood, spleen and bone marrow of tumor-bearing mice. Representative flow cytometry bar plots show: a the percentage of blood, spleen and bone marrow PMN-MDSCs in TuBo ( n = 8) and 4T1 ( n = 7/blood and bone marrow; n = 9/spleen) mock and p140Cap tumor-bearing mice; ( b ) the percentage of lung PMN-MDSCs in 4T1 mock and p140Cap tumor-bearing mice ( n = 5 mice/group; data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test). c G-CSF protein levels in sera of tumor-bearing mice. G-CSF protein levels were measured in sera collected from TuBo ( n = 4) and 4T1 ( n = 5) mock and p140Cap tumor-bearing mice by ELISA (Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test).

Article Snippet: The following antibodies were used (at a dilution of 1:200): CD45-VioGreen (Cat#130-110-803), CD11b-FITC (Cat#130-110-803), Ly6G-VioBlue (Cat#130-119-986), Ly6C-APC-Vio770 (Cat#130-121-439), F4/80-PE-Vio770 (Cat#130-118-320), MHC-II-APC (Cat#130-102-139), CD3-FITC (Cat#130-119-135), CD4-APC-Vio770 (Cat#130-119-134), CD8-VioBlue (Cat#130-123-865), CD49b-PE (Cat#130-123-702) all from Miltenyi Biotec (Miltenyi Biotec B.V. & Co. Bologna, Italy), while CD206-PE (Cat#141706) was from BioLegend (BioLegend, San Diego CA, USA).

Techniques: Flow Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The Culture Dish Surface Influences the Phenotype and Dissociation Strategy in Distinct Mouse Macrophage Populations

doi: 10.3389/fimmu.2022.920232

Figure Lengend Snippet: Detaching approaches were performed with two types of BMDMs. (A) Schematic diagram of experimental workflow. Post 7 days’ maturation, GM and M were dissociated by enzymic or nonenzymic methods with pipetting or scraping as shown in Table 1 . The percentage of subpopulations was detected by flow cytometry. Dead cells were further discarded using gradient centrifugation for GM-TC samples when more viable GM-TC cells were needed. (B) Percentages of live BMDMs (n = 4 for each group) using different dissociation methods without dead cell removal process. (C, H) Gating strategies for GM- (C) and M- (H) BMDMs cultured on TC dishes and dissociated with accutase followed by necessary mechanical steps in flow cytometry. BMDMs were detached, blocked and stained with the indicated antibodies. Cells were gated for single cells, followed by gating live cells (7AAD-) and CD11b+ populations. The live 7AAD- CD11b+ cells were further analyzed for the specific subpopulations. (C) P1: CD11b + MHCII hi , P2: CD11b hi MHCII + , P3: CD11b hi/+ MHCII low/- , and CD11b + Ly6C + cells; (H) P1: CD11b + MHCII hi/+ , P2: CD11b + MHCII low/- , and CD11b + Ly6C + cells. (D–G) The percentages of CD11b hi MHCII + (D) , CD11b hi/+ MHCII low/- (E) , CD11b + Ly6C + (F) and CD11b + MHCII hi (G) subpopulations in GM-TC and GM-noTC samples. (I, J) The percentages of CD11b + MHCII low/- (I) and CD11b + MHCII hi/+ (J) subpopulations in M-TC and M-noTC samples. (K–N) MFI values of F4/80 (K) , MHCII (L) , CD11b (M) and Ly6C (N) in the CD11b+ cells of GM and M. (O) MFI values of CD11b, F4/80, MHCII and Ly6C in the CD11b+ cells of GM-TC samples dissociated with EDTA (control group) or accutase at different temperatures and treatment times (n = 4 for each group). RT_10 min means incubation with accutase for 10 min at RT, RT_5 min +37°C_5 min means incubation for 5 min at RT followed by 5 min at 37°C, and RT_5 min+37°C_10 min means incubation for 5 min at RT followed by 10 min at 37°C. (P) Cell morphology of GM and M cultured on TC and noTC dishes. Scale bars, 100 μm. (Q–S) GM and M cells were treated with LPS (100 ng/ml) for 3 h. Total RNA was prepared and used to determine relative mRNA levels of proinflammatory factors IL1β (Q) , TNFα (R) and IL6 (S) . One-way ANOVA was performed for (D–G) and (I, J) . Two-way ANOVA was performed for (B) , (K–O) and (Q-S) . For (B) , P < 0.05 is indicated by * for the comparison of other 3 dissociation methods vs the group with the lowest cell viability in each type of BMDMs. For (D-G) , (I, J) and (K–N) , P < 0.05 is indicated by * for the comparison of other 3 dissociation methods vs PBS groups within GM-TC, GM-noTC, M-TC and M-noTC samples, and # for comparison of the accutase dissociated samples between GM-TC vs GM-noTC or M-TC vs M-noTC. For (O) , P < 0.05 is indicated by * for the comparison of accutase treated samples with EDTA treated ones. For (Q–S) , P < 0.05 is indicated by * for the comparison of the TC vs noTC samples under the same conditions.

Article Snippet: Debris Removal Solution (Cat. 130-110-203) and antibodies against Ly6C (APC) (Cat. 130-111-779) were from Miltenyi Biotec.

Techniques: Flow Cytometry, Gradient Centrifugation, Cell Culture, Staining, Control, Incubation, Comparison

Journal: Frontiers in Endocrinology

Article Title: Myeloid Wls expression is dispensable for skin wound healing and blood vessel regeneration

doi: 10.3389/fendo.2022.957833

Figure Lengend Snippet: Myeloid Wntless (Wls) deletion does not have significant impact on the endothelial hierarchy and the myeloid populations during skin homeostasis. (A) Representative flow cytometry plots and gating strategy used to identify myeloid subpopulations in the skin of Wls fl/fl LysM-Cre + and Wls fl/fl LysM-Cre neg mice. Granulocytes were gated as CD11b + Ly6G + cells (gate i). CD11b + Ly6G neg cells (gate ii) were subsequently gated into Ly6C + MHCII neg monocytes (gate iii), Ly6C + MHCII + monocytes/Mϕ (gate iv), Ly6C neg MHCII + Mϕ (gate v) and Ly6C neg MHCII neg cells (gate vi). (B) Percentage of live CD45 + (i) granulocytes and CD11b + Ly6G neg myeloid cells consisting of (ii) monocytes, monocytes/Mϕ, Mϕ and Ly6C neg MHCII neg cells of Wls fl/fl LysM-Cre + and Wls fl/fl LysM-Cre neg (Control) mice. (C) Representative flow cytometry analysis of the endothelial hierarchy in the skin of Wls fl/fl LysM-Cre + and Wls fl/fl LysM-Cre neg mice during homeostasis. (C) CD144 + Lin neg cells (gate i) consists of CD31 + CD34 + D (gate ii), CD31 neg CD34 + EVP (gate iii) and CD31 neg CD34 neg M (gate iv) cells. (D) Percentage of live (i) CD144 + Lin neg cells and (ii) EVPs, D and M cells in the skin of Wls fl/fl LysM-Cre + and the Wls fl/fl LysM-Cre neg control littermates. No difference was observed in the total percentage of CD144 + LIN neg endothelial cells, as well as the EVP, D and M between the two groups. Data are represented as mean ± SD. Statistical analysis was performed using unpaired Student t-tests. ns= not significant. Data are representative of 4 individual animal samples.

Article Snippet: Myeloid cells phenotyping was performed as above by staining 1 x 10 6 cells with rat anti-mouse F4/80-BV421 (clone T45-2342) (BD Bioscience, Cat# 565411, RRID: AB_2734779), rat anti-mouse CD11b-BV785 (clone M1/70) (BioLegend, California, USA, Cat# 101243, RRID: AB_2561373), rat anti-mouse Ly6G-PE (clone 1A8) (BD Bioscience, Cat# 551461, RRID: AB_394208), rat anti-mouse Ly6C-APC.Cy7 (clone AL-21) (BD Bioscience, Cat# 560596, RRID: AB_1727555), rat anti-mouse MHCII-FITC (cloneM5/114.15.2) (BioLegend, Cat# 107606, RRID: AB_313321), and rat anti-mouse CD45-BUV395 (clone 30-F11) (BD Bioscience, Cat# 564279, RRID: AB_2651134).

Techniques: Flow Cytometry

Journal: Frontiers in Endocrinology

Article Title: Myeloid Wls expression is dispensable for skin wound healing and blood vessel regeneration

doi: 10.3389/fendo.2022.957833

Figure Lengend Snippet: Inflammatory response in Wls fl/fl LysM-Cre + mice remain unperturbed after wounding. (A) Schematic of the experimental timeline demonstrating the Wls fl/fl LysM-Cre wound healing time course. Two 6mm wounds were created on the murine dorsal skin using punch biopsy at day 0. Granulation tissues were collected at day 1, 3, 5, and 7 for flow cytometry and immunofluorescence analysis. (B) Representative flow plots showing the gating strategy used to identified granulocytes (gate i), CD11b + Ly6G neg (gate ii) cells which are further subdivided into monocytes (gate iii), monocyte/Mϕ (gate iv), Mϕ (gate v) and Ly6C neg MHCII neg cells (gate vi). (C) Flow cytometry analysis comparing granulation of Wls fl/fl LysM-Cre + and Wls fl/fl LysM-Cre neg mice enriched in (i) granulocytes and (ii) CD11b + Ly6G neg myeloid cells, (iii) monocytes, (iv) monocytes/Mϕ, (v) Mϕ, and (vi) Ly6C neg MHCII neg cells at all time points post wounding. Highest number of monocytes were recorded at day 1 and its percentage reduces progressively between day 3 and day 7. Monocytes/Mϕ peaked at day 3. Wls fl/fl LysM-Cre + has slightly higher percentage of Mϕ at day 5 (p = 0.08). Ly6C neg MHCII neg peaked at day 3 during the inflammatory stage of the wound healing with no difference seen between the Wls fl/fl LysM-Cre + and Wls fl/fl LysM-Cre neg mice. Data are represented as mean ± SD. Statistical analysis was performed using unpaired Student t-tests. ns, not significant. Data are representative of 3-4 individual animal samples per time point.

Article Snippet: Myeloid cells phenotyping was performed as above by staining 1 x 10 6 cells with rat anti-mouse F4/80-BV421 (clone T45-2342) (BD Bioscience, Cat# 565411, RRID: AB_2734779), rat anti-mouse CD11b-BV785 (clone M1/70) (BioLegend, California, USA, Cat# 101243, RRID: AB_2561373), rat anti-mouse Ly6G-PE (clone 1A8) (BD Bioscience, Cat# 551461, RRID: AB_394208), rat anti-mouse Ly6C-APC.Cy7 (clone AL-21) (BD Bioscience, Cat# 560596, RRID: AB_1727555), rat anti-mouse MHCII-FITC (cloneM5/114.15.2) (BioLegend, Cat# 107606, RRID: AB_313321), and rat anti-mouse CD45-BUV395 (clone 30-F11) (BD Bioscience, Cat# 564279, RRID: AB_2651134).

Techniques: Flow Cytometry, Immunofluorescence