magnetic activated cell sorting macs buffer  (Miltenyi Biotec)

Journal: Science Advances

Article Title: Combined innate immune cell therapy, tumor-specific antibody, and radiation prompt antitumor response in pancreatic cancer models

doi: 10.1126/sciadv.adx2984

Figure Lengend Snippet: ( A ) Schematic representation for the preparation of the cell product (ICP IL2ZA ) from spleens of tumor-bearing C57BL/6 mice. ( B ) Flow cytometry of mouse splenocytes before and after TCRαβ depletion (ICP). ( C and D ) Increased expression of CD16 + and IFN-γ + in monocytes (C) GRANZYME B + (GZMB + ) and Perforin + in NK cells (D) after overnight stimulation with IL2ZA stimulation (ICP IL2ZA ). ( E and F ) Elevated expression of CD45 − annexin V + (AVS + ) or annexin V + cells detected by flow cytometry (E) or Incucyte (F) when Panc02ER cells were cocultured with ICP IL2ZA in presence of cetuximab (Cet). ( G and H ) Expression of the NKG2D ligands RAE and ULBP1 in Panc02ER cells at the indicated time after 4-Gy radiation (RT). ( I ) Elevated annexin V + detected by Incucyte assay of Panc02ER cells after 4-Gy RT, cell therapy (ICP IL2ZA ), and cetuximab treatment. ( J ) Preparation of ICP IL2ZA from mouse splenocytes for in vivo study. ( K ) Mice bearing Panc02ER tumor were treated with 4-Gy RT at days (d) 0, 7, and 14; cetuximab at days 1, 8, and 15; and ICP IL2ZA at days 2, 9, and 16. ( L to N ) The resulting mean tumor volume ± SD and individual tumor volumes (L), survival (M), and body weights (N) are shown ( n = 8 to 15 per group). Combination treatment resulted in slowed tumor growth and the greatest improvement in survival. Comparisons between groups were based on the average growth volume (the area under the growth curve divided by the follow-up period) . Kaplan-Meier curves were used to estimate the survival probabilities and were compared between groups by the log-rank test. Unpaired t test was performed for (C) to (F). Significance is demarcated by an asterisk, with the color of the asterisk representing the group from which the sample is significantly different; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For isolation of the NK or CD14 cells from the PBMCs, hICP or hICP IL2ZA magnetic-activated cell sorting (MACS)–cell separation microbeads and isolation kit (130-092-657 and 130-096-537, Miltenyi Biotec) were used according to the manufacturer’s instruction.

Techniques: Flow Cytometry, Expressing, In Vivo

Journal: Science Advances

Article Title: Combined innate immune cell therapy, tumor-specific antibody, and radiation prompt antitumor response in pancreatic cancer models

doi: 10.1126/sciadv.adx2984

Figure Lengend Snippet: ( A ) Panc02ER tumor–bearing mice were treated with 4-Gy RT at day 0, cetuximab at day 1, and ICP IL2ZA at day 2. The treatment cycle repeated starting at day 7. Tumor, TDLN, and blood were collected at day 16 for immune profiling. ( B ) Tumor-infiltrating CD8 and NK cells were detected by IHC ( n = 3). Scale bar, 200 μm. ( C to G ) Immune cell markers in tumors of mice receiving PBS control or RT, cetuximab, and ICP IL2ZA (3xTx) ( n = 5 animals per cohort). (C) Percentage of CD3 − CD45 + NK1.1 + and IFN-γ + NK cells. (D and E) Percentage of CD80 + CD206 − CD11b + F4/80 + macrophages (Mac-pro) (D) and DCs and mean fluorescence intensity (MFI) of CD80 + DC cells (E). (F) Percentages of CD8 + T (CD45 + CD3 + CD8 + ) cells and MFI of activation markers (IFN-γ and CD69 + ) and exhaustion markers (PD-1 + and CD25 + FOXP3 + ). (G) Percentages of CD4 + T cells (CD45 + CD3 + CD4 + ) and MFI of activation (IFN-γ + and CD69 + ) and exhaustion markers (PD-1 + and CD25 + FOXP3 + ) ( n = 5). ( H to L ) Immune cell markers from TDLN (H) and percentage of NK cells and TNFα + NK cells. (I and J) Percentage of CD80 + CD206 − CD11b + F4/80 + macrophages and ratio of CD80 + CD206 − CD11b + F4/80 + to CD206 + CD80 − (Mac-sup) macrophages and percentage of DC (CD45 + CD11c + MHCII + ) and DC80 percent among the parent DCs. (K and L) Percentage of central memory T cells (T cm cells) (CD44 + CD62L + ) and MFI of activated (CD69 + ) cells among CD8 + (K) and CD4 + cells (L). ( M to O ) Immune cell markers in blood are percentage of CD8 + and IFN-γ + CD8 + cells in the blood (M), percentage of CD4 + and its T H 1 + markers (IFN-γ, TBET, and TNFα) (N), and NK cells and NKG2D + NK cells (O). Statistical significance was calculated via t test in (C) to (O). Data are presented as means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For isolation of the NK or CD14 cells from the PBMCs, hICP or hICP IL2ZA magnetic-activated cell sorting (MACS)–cell separation microbeads and isolation kit (130-092-657 and 130-096-537, Miltenyi Biotec) were used according to the manufacturer’s instruction.

Techniques: Control, Fluorescence, Activation Assay

Journal: Science Advances

Article Title: Combined innate immune cell therapy, tumor-specific antibody, and radiation prompt antitumor response in pancreatic cancer models

doi: 10.1126/sciadv.adx2984

Figure Lengend Snippet: ( A ) Schematic representation for the of preparation of hICP from PBMC. ( B ) Flow cytometry analysis before and after TCRαβ cells depletion. ( C ) Depletion of TCRαβ enrich CD14 + (monocytes) and CD56 + (NK) cells ( n = 3 donors). Gating strategy (left) and percentage of indicated cell types (right). ( D and E ) (D) Depleted TCRαβ cells (hICP) were stimulated with IL-2 and ZA for 16 hours, and (E) CD16 expression was analyzed by flow cytometry (left) and quantified (right). ( F ) GRANZYME B and Perforin were analyzed after treatment with PBS, IL-2, ZA, or IL2ZA ( n = 4 donors). ( G ) Release of IFN-γ and TNFα measured by enzyme-linked immunosorbent assay (ELISA). Statistical significance was calculated via t test or ANOVA; means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For isolation of the NK or CD14 cells from the PBMCs, hICP or hICP IL2ZA magnetic-activated cell sorting (MACS)–cell separation microbeads and isolation kit (130-092-657 and 130-096-537, Miltenyi Biotec) were used according to the manufacturer’s instruction.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Combined innate immune cell therapy, tumor-specific antibody, and radiation prompt antitumor response in pancreatic cancer models

doi: 10.1126/sciadv.adx2984

Figure Lengend Snippet: ( A ) Percentage of live cells after IL2ZA-stimulated PBMC (PBMC IL2ZA ) or hICP IL2ZA were cocultured with red fluorescent protein (RFP)–transduced PANC1 cells and cetuximab at a 10:1 effector-to-target cell ratio (E:T). ( B ) Flow cytometry analysis of regulatory T (CD4 + FOXP3 + ) cells in PBMC IL2ZA or hICP IL2ZA . ( C ) Percentage of live cells after PANC1 cells were cocultured with NK cells from either PBMC IL2ZA or hICP IL2ZA . ( D ) MFI of TNFα and IFN-γ CD56 + NK cells isolated from PBMC IL2ZA or hICP IL2ZA cocultured with PANC1 cells and cetuximab. ( E ) Experimental schema (top) and example of effector cell (green) infiltration of a PANC1 (red) spheroid (bottom). ( F ) Top: Schema depicting coculture of PANC1 cells with hICP IL2ZA , NK-depleted hICP IL2ZA (hICP IL2ZA-NK ), or CD14-depleted hICP IL2ZA (hICP IL2ZA-CD14 ). Control or NK- or CD14-depleted effector cells were prepared before IL2ZA stimulation. Bottom: Percentage of live PANC1 cells after coculture with effector cells and cetuximab. ( G ) Monocytes (CD14 + ) were isolated from either PBMC (CD14 PBMC ) or hICP IL2ZA (CD14 IL2ZA ) and then cocultured with treatment-naïve NK cells for 16 hours ( n = 2 donors). Schema (top) and percentage of CD16 + and MFI of IFN-γ + among NK cells (CD56 + ) after coculture with indicated CD14 cells (bottom). ( H ) Experimental schema (top) and percentage of NK cells (CD56 + ) and IFN-γ + NK cells and concentration of secreted IFN-γ and TNFα in medium after 16 hours of coculture are shown (bottom). PANC1 cells were cocultured with either hICP IL2ZA or hICP IL2ZA-CD14 and cetuximab. ( I ) Polyfunctional strength index of CD14 monocytes and NK cells from PBMC (CD14 PBMC and NK PBMC ) and hICP IL2ZA (CD14 IL2ZA and NK IL2ZA ). Data represent the mean value. ( J ) Individual t-SNE plot of IFN-γ, GRANZYME B, IL-7, Perforin, GM-CSF, IL-8, IL-9, and TNFα in CD14 + and NK cells from PBMC and hICP IL2ZA . Statistical significance was calculated via ANOVA or t test; means ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For isolation of the NK or CD14 cells from the PBMCs, hICP or hICP IL2ZA magnetic-activated cell sorting (MACS)–cell separation microbeads and isolation kit (130-092-657 and 130-096-537, Miltenyi Biotec) were used according to the manufacturer’s instruction.

Techniques: Flow Cytometry, Isolation, Control, Concentration Assay

Journal: Science Advances

Article Title: Combined innate immune cell therapy, tumor-specific antibody, and radiation prompt antitumor response in pancreatic cancer models

doi: 10.1126/sciadv.adx2984

Figure Lengend Snippet: ( A ) Percentage live PANC1, MIA PaCa, T3M4, or Capan2 after coculture with hICP IL2ZA , mAb [cetuximab or trastuzumab (Trast)], or combined hICP IL2ZA + mAb. E:T cell ratio was 10:1. ( B ) Cytotoxicity analysis of RFP-transduced PANC1 spheroids after indicated coculture. hr, hours. ( C ) Percentage of PANC1 cells expressing MHCI chain-related protein A and B (MICA and MICB) over time after radiation (RT) with 4 Gy. ( D ) Percentage of live PANC1 cells after the indicated treatments. ( E to M ) (E) Treatment schema for NRG mice bearing PANC1 tumors. (F to I) Average (F) and individual (G) tumor volume, survival (H), and weight (I) of mice in the cetuximab group receiving the indicated treatment. (J to M) Average (J) and individual (K) tumor volume, survival (L), and weight (M) of mice in the trastuzumab group receiving the indicated treatment. ( n = 5 to 10 mice per groups). ( N and O ) Treatment schema [(N), top] for hICP IL2ZA tracking in PANC1 tumor–bearing mice treated with 1 cycle of combination therapy. Alexa Fluor 647–tagged anti-huCD45 + antibody was injected intravenously 24 hours after of hICP IL2ZA , followed by IVIS imaging 24 hours later. hICP IL2ZA accumulation in PANC1 tumors was imaged for five mice per group receiving the indicated treatment [(N), bottom]. (O) Percentage of MICA/B + -expressing cells among CD45 − live cells in PANC1 tumors. Statistical significance was calculated in (A) and (C) via ANOVA with Tukey’s multiple comparison test. t test was performed for (D). Tumor volume and survival probabilities were compared between groups by average tumor growth volume, i.e., the area under the growth curve divided by the follow-up period and log-rank test. Significance is demarcated by asterisk, with the color of the asterisk representing the group from which the sample is significantly different; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For isolation of the NK or CD14 cells from the PBMCs, hICP or hICP IL2ZA magnetic-activated cell sorting (MACS)–cell separation microbeads and isolation kit (130-092-657 and 130-096-537, Miltenyi Biotec) were used according to the manufacturer’s instruction.

Techniques: Expressing, Injection, Imaging, Comparison