medium per well  (Thermo Fisher)

Journal: iScience

Article Title: Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design

doi: 10.1016/j.isci.2023.107952

Figure Lengend Snippet: Profile OC tumor cells and evaluate cell-based immunotherapy (CBI) strategies (A) Experimental design to collect and study tumor and immune cells from primary OC patient samples. (B) Flow cytometry showing the OC patient sample-derived tumor cells. Tumor cells were identified as non-immune/fibroblast/endothelial (CD45-FAP-CD31 − ) cells. FAP, fibroblast activation protein. (C) Table showing the antigen expression level on tumor cells from 26 OC samples. CAR, chimeric antigen receptor; MSLN, mesothelin; MUC16, mucin-16; ESO, esophageal squamous cell carcinoma; NY-ESO-1, New York ESO-1; NK, natural killer cell; MICA/B, MHC class I chain related-proteins A and B; ULBP, UL16 binding protein. (D) Quantification of tumor antigen expressions from the 26 OC samples. (E and F) MSLN expression on tumor cells and MSLN-targeting CAR-engineered T (MCAR-T) cell targeting. (E) FACS analyses of MSLN expression on tumor cells from three OC patient samples. MSLN high-, medium-, and low-expressing tumor samples were presented. (F) OC tumor cell killing data by MCAR-T cells at 24 h (E:T = 1:1, n = 3). - MCAR-T, no MCAR-T cells co-cultured with tumor cells. (G-H) NY-ESO-1 expression on tumor cells and ESO TCR-engineered T (ESO-T) cell targeting. (G) FACS analyses of NY-ESO-1 expression on tumor cells from three OC patient samples. NY-ESO-1 high-, medium-, and low-expressing tumor samples were presented. (H) OC tumor cell killing data by ESO-T cells at 24 h (E:T = 2:1, n = 3). - ESO-T, no ESO-T cells co-cultured with tumor cells. (I) OC tumor cell killing data by PBMC-derived NK cells at 24 h (E:T = 5:1, n = 3). - NK, no NK cells co-cultured with tumor cells. (J and K) FACS analyses of cancer stem cell (CSC) features of tumor cells from 26 OC samples. Transcription factors Oct3/4. SOX2, and Nanog were used as OC CSC markers. (J) SPICE analysis of CSC marker expression from 3 pairs of matched OC samples. Pie charts reflect proportions of indicated tumor cell groups expressing indicated numbers (0–3) of CSC markers. Colored arcs indicate the specific combinations of CSC markers expressed. (K) Summary of OC patient samples categorized based on the expression level of CSC markers. The ratios above the bar represent the number of marker-positive samples versus the total number of samples. Representative of 1 (C, D, J, and K) and >20 (A, B, and E-I) experiments. Data are presented as the mean ± SEM. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, by Student’s t test.

Article Snippet: On day 14, cells were harvested, counted, and reseeded into an LDCM-coated 12-well plate in 1 mL of T cell progenitor maturation medium per well (TPMM, StemCell Technologies) (5 x 10 5 - 1 x 10 6 cells/well).

Techniques: Flow Cytometry, Derivative Assay, Activation Assay, Expressing, Binding Assay, Cell Culture, Marker

Journal: iScience

Article Title: Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design

doi: 10.1016/j.isci.2023.107952

Figure Lengend Snippet: Identify CD1d as an OC TME biomarker and assess TME targeting strategy via iNKT cells (A–C) FACS analyses of CD1d expression on the indicated immune cells. (A) CD1d expression on T, B, NK, and TAM/MDSC cells. Data from sample #8 and #19 were shown. (B) Quantification of CD1d expression on different immune cells (n = 26). (C) Quantification of CD1d expression on TAMs between chemonaive and recurrent samples. Total 26 OC samples were shown. (D-I) Studying OC TME targeting by Allo HSC-iNKT cells. Healthy donor peripheral blood mononuclear cell-derived αβ T (PBMC-T) cells were included as a therapeutic cell control. (D) Experimental design. (E and F) FACS analyses of live CD11b + CD1d + cells 24 h after co-culturing with Allo HSC-iNKT or PBMC-T cells. Data of chemonaive sample #4 (E) and recurrent sample #23R (F) were shown. (G) TAM/MDSC killing data by Allo HSC-iNKT and PBMC-T cells at 24 h (n = 3). (H) TAM/MDSC killing efficacy of Allo HSC-iNKT cells on chemonaive and recurrent OC samples. Data from a total 26 OC samples were shown. (I) T, B, and NK cell killing data by Allo HSC-iNKT and PBMC-T cells at 24 h (n = 3). Results from OC samples with a high proportion of B and NK cells were presented to ensure sufficient cell numbers for analysis. (J and K) Studying CD1d-mediated OC TME targeting by Allo HSC-iNKT cells. Anti-CD1d antibody was added into the coculture to block CD1d/iNKT TCR recognition. (J) Experimental design. (K) TAM/MDSC killing data by Allo HSC-iNKT cells at 24 h (n = 3). (L-M) Studying the tumor cell killing efficacy and mechanisms of Allo HSC-iNKT cells. (L) Tumor cell killing data of OC tumor cells by Allo HSC-iNKT cells at 24 h (E:T = 2:1, n = 3). Healthy donor-derived T and B cells were included as target cell controls. (M) Tumor cell killing data at 24 h (E:T = 2:1, n = 3). Anti-NKG2D and/or anti-DNAM-1 antibodies were added into the coculture to block NK receptor/ligand recognition. Representative of 1 (B, C, G-I, and L) and >20 (A, D-F, J, K, and M) experiments. Data are presented as the mean ± SEM. ns, not significant, ∗∗p < 0.01, ∗∗∗p < 0.001, by Student’s t test (B right, C, and H), and by one-way ANOVA (B left, K, and M).

Article Snippet: On day 14, cells were harvested, counted, and reseeded into an LDCM-coated 12-well plate in 1 mL of T cell progenitor maturation medium per well (TPMM, StemCell Technologies) (5 x 10 5 - 1 x 10 6 cells/well).

Techniques: Biomarker Discovery, Expressing, Derivative Assay, Control, Blocking Assay

Journal: iScience

Article Title: Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design

doi: 10.1016/j.isci.2023.107952

Figure Lengend Snippet: Summary of the OC tumor cell and TME changes during OC disease progression, and the roadmap to design the cell-based immunotherapies

Article Snippet: On day 14, cells were harvested, counted, and reseeded into an LDCM-coated 12-well plate in 1 mL of T cell progenitor maturation medium per well (TPMM, StemCell Technologies) (5 x 10 5 - 1 x 10 6 cells/well).

Techniques: Biomarker Discovery

Journal: iScience

Article Title: Profiling ovarian cancer tumor and microenvironment during disease progression for cell-based immunotherapy design

doi: 10.1016/j.isci.2023.107952

Figure Lengend Snippet:

Article Snippet: On day 14, cells were harvested, counted, and reseeded into an LDCM-coated 12-well plate in 1 mL of T cell progenitor maturation medium per well (TPMM, StemCell Technologies) (5 x 10 5 - 1 x 10 6 cells/well).

Techniques: Purification, Control, Blocking Assay, Activation Assay, Virus, Recombinant, Cell Culture, Saline, Cell Isolation, Staining, Transfection, Plasmid Preparation, Software

Journal: PLoS ONE

Article Title: Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

doi: 10.1371/journal.pone.0044371

Figure Lengend Snippet: A large decrease in hippocampal-derived neurospheres was observed in PRL −/− mice compared to wild-type females (***p<0.001; n = 3 PRL +/+ and 6 PRL −/− animals) and males (***p<0.001; n = 9 animals each). There was also a significant decrease in hippocampal-derived neurosphere number in PRL +/− compared to wild-type females (***p<0.001; n = 3 PRL +/+ and 5 PRL +/− animals) and males (***p<0.001; n = 6 PRL +/+ and 7 PRL +/− animals).

Article Snippet: Adult hippocampal or SVZ tissue was dissected and dissociated, as described above, and the cells were plated at a density of approximately one hippocampus or SVZ per 96-well plate (Falcon/BD Biosciences) with 200 µl neurosphere medium per well.

Techniques: Derivative Assay