transwell tc inserts  (Sarstedt)

Journal: Scientific Reports

Article Title: Preclinical evaluation of DC-CIK cells as potentially effective immunotherapy model for the treatment of glioblastoma

doi: 10.1038/s41598-024-84284-5

Figure Lengend Snippet: ( A ) Schematic of 2D single-cell and 3D spheroid models for co-culturing DC-CIK and GBM cells in a transwell system. ( B ) BTSC233 (stem cells and differentiated cells) were cultured at a 10:1 effector: target ratio for 24 h comparing regular cytotoxicity and transwell cytotoxicity. The cytotoxic effect of DC-CIK cells was measured by flow cytometry. ( C ) DC-CIK cells on the apoptosis of GBM cell lines (84 and G35) by the Flow Cytometry assay. Flow cytometry figure of changes in the proportion of early apoptosis cells and late apoptosis or necrosis cells. Cells were stained with FITC Annexin V and Percp 7AAD. The result is one of the representative data. ( D ) IFN-γ and TNF-α secretion in DC-CIK cells targeting GBM cell lines 84, G35, and BTSC233. ( E ) Relative mRNA expression changes of Wnt/beta-catenin pathway-related genes on BTSC233 (spheroid) cell line after coculturing with DC-CIK cells (E: T ratio 1:1) for 24 h. The results represent data from three separate experiments and are presented as mean ± SD. Significance levels were determined using two-way ANOVA with Bonferroni’s post-hoc test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Co-culturing with transwell system (TC-insert, pore size 0.4 μm, Sarstedt AG & Co. KG, Nümbrecht, Germany) was performed at E: T ratio 10:1 with BTSC233 and GBOs for up to 72 h.

Techniques: Cell Culture, Flow Cytometry, Staining, Expressing

Journal: Scientific Reports

Article Title: Preclinical evaluation of DC-CIK cells as potentially effective immunotherapy model for the treatment of glioblastoma

doi: 10.1038/s41598-024-84284-5

Figure Lengend Snippet: ( A ) Diagram outlining the steps to prepare and co-culture DC-CIK cells with GBOs with downstream analyses. The medium and culture conditions are indicated below each step. CM, CIK cell medium; GM, GBO medium; RH, relative humidity. ( B ) Phase contrast images of individual GBOs at 0, 24, 48, and 72 h, co-cultured with DC-CIK cells with or without a transwell system, and a control group of GBOs alone. Representative images are based on the analysis of 10 GBOs. Scale bar, 500 μm. ( C ) Sample phase contrast images of the edge of GBOs when transwell co-cultured with DC-CIK cells for 72 h (E: T 10:1 W/T transwell E: T 1:1 W/O transwell). Representative images are based on the analysis of 10 GBOs. ( D ) Quantification of IFN-γ in media from co-cultures with DC-CIK cells, with or without a transwell system, was performed using ELISA at 0, 24, 48, and 72 h. Values represent mean ± s.e.m. ( n = 3 GBOs per sample per timepoint). ( E ) Sample confocal images of fluorescent immunohistology for cleaved caspase 3 (CC3) and cluster of differentiation 3 (CD3) in GBOs after co-culture with either CIK cells for 48 h (E: T 1:5), showing increased apoptosis when co-cultured with CIK cells, with higher-magnification images of boxed regions highlighting apoptotic cells near CIK cells at the GBO periphery and core. Representative images are based on the analysis of five GBOs. Images were taken using Zen 2 Blue software and were processed using Illustrator CC, ImageJ, Imaris and/or Photoshop CC software.

Article Snippet: Co-culturing with transwell system (TC-insert, pore size 0.4 μm, Sarstedt AG & Co. KG, Nümbrecht, Germany) was performed at E: T ratio 10:1 with BTSC233 and GBOs for up to 72 h.

Techniques: Co-Culture Assay, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Software

Journal: Frontiers in Immunology

Article Title: 1,25-dihydroxyvitamin-D 3 distinctly impacts the paracrine and cell-to-cell contact interactions between hPDL-MSCs and CD4 + T lymphocytes

doi: 10.3389/fimmu.2024.1448597

Figure Lengend Snippet: In the direct co-culture model without inserts, 1,25(OH) 2 D 3 differently impacts CD4 + T lymphocyte proliferation and viability via hPDL-MSCs compared to the other models. Depending on the co-culture model (A) , hPDL-MSCs were seeded in 6-well plates or at the membrane’s underside of transwell inserts (TC) and were treated with 1,25(OH) 2 D 3 . CD4 + T lymphocytes were added either into the TC insert or directly to the hPDL-MSCs and were activated by phytohemagglutinin. After five days of incubation, CFSE and Pi stainings were analyzed by flow cytometry to determine the proliferation and viability of CD4 + T lymphocytes, respectively (B-I) . The % of original CD4 + T lymphocytes that have proliferated is presented in (B) , whereas the % of (Pi + ) dead CD4 + T lymphocytes is shown in (C) . For the undivided (UD) and each divided (G1-G6) generation, the number of original CD4 + T lymphocytes is demonstrated as the percentage of the total original cell number (D-F) . (G, H) show representative histograms with absolute numbers of CD4 + T lymphocytes for each generation (UD and G1-G6). The data were obtained from five independent repetitions using hPDL-MSCs isolated from five distinct individuals. In (B, C) , single data points, the median, and the confidence intervals (95%) are presented. The data in (D-F) are presented as box-whisker plots, including the minimum and maximum values. Statistically significant differences are shown by providing P-values obtained using the Wilcoxon Test for pairwise comparison. Figure 1A was created with Biorender .

Article Snippet: After an additional 48 hours of incubation, 1x10 6 freshly isolated and CFSE-stained CD4 + T lymphocytes were added in transwell (TC) inserts (pore size: 0.4 μm; Sarstedt, Biedermannsdorf, Austria) using 1 ml fully supplemented RPMI-1640 medium per insert.

Techniques: Co-Culture Assay, Incubation, Flow Cytometry, Isolation, Whisker Assay, Comparison