Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Closed-system manufacturing of therapeutic NK cells using automated cell enrichment and concentration processes enables scalable, robust and cost-effective solutions

doi: 10.3389/fbioe.2025.1586912

Figure Lengend Snippet: NK drug substance composition before and after the concentration process using the CliniMACS Prodigy ® . (A) Total CD45 + cell (identified as CD45 + living) number before and after the cell concentration process (n = 29). (B) Total CD45 + cell yield after the cell concentration process, according to low (n = 7), medium (n = 14) or high (n = 8) cell culture volume. (C) CD56 + NK cell (identified as CD45 + CD56 + CD3 − living) purity before and after cell concentration process (n = 29). (D) CD56 + cell purity after cell concentration process, according to low (n = 7), medium (n = 14) or high (n = 8) cell culture volume. (E) CD19 + B cell impurity content (identified as CD45 + CD19 + living) before and after cell concentration process (n = 7). For all plots, the central line denotes the median; boxes delimitate the 25th and 75th percentiles and whiskers mark the minimum and maximum. For statistical analysis, paired T-test was used in (A, C, E) , one-way ANOVA in (B,D) .

Article Snippet: CD19 , PE , LT19 , 170-081-057 , Miltenyi , GMP 1-22, R&D 25-36.

Techniques: Concentration Assay, Cell Culture

Journal: Molecular therapy. Methods & clinical development

Article Title: Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus.

doi: 10.1016/j.omtm.2022.05.006

Figure Lengend Snippet: Figure 3. Immunophenotyping of 1D3-CD19CAR- GFP (blue, circle), FMC63-CD19CAR-GFP (red, square), or control (GFP only, green, triangle) trans- duced T cells in peripheral blood of treated mice Values shown are the percentage of total CD3+, CD90.2+

Article Snippet: In order to verify the ability for both the 1D3-and FMC63-CD19CAR to target the mouse CD19 antigen, the A20 cells were incubated with 10, 1, 0.1, 0.001, or 0 mg/mL of either the 1D3CD19-blocking antibody (152402, BioLegend) or the FMC63-CD19- Molecular Th blocking antibody (NBP2-52716, Novus Biologicals).

Techniques: Control

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: (A) Representative flow cytometric histograms showing the specificity of dimeric CD20 aptamer WB1/1.CD20.1_3S across CD20-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (B) Bar graph quantifying the mean fluorescence intensities of WB1/1.CD20.1_3S binding, highlighting significant specificity (*p < 0.0001). (C; E) Fluorescence histograms for CD19 homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (D; F) Quantification of homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, binding specificity using mean fluorescence intensity. (G) Fluorescence histograms for CD19 heterodimeric aptamer WB15/17.CD19.1_3S, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (H) Quantification of heterodimeric aptamer WB15/17.CD19.1_3S binding specificity using mean fluorescence intensity. (I-L) Confocal microscopy images showing colocalization of bivalent WB1/1.CD20.1_3S (L1-L2: Cy3, RED) with a CD20-specific antibody (J1-J2: APC, GREEN) on Raji cells. DNA random aptamer controls and isotype antibody controls confirmed specificity. The aptamer binds to the cell surface membrane, as shown in zoomed-in views (L1-L2). Panel M-P: Confocal microscopy images of bivalent WB17/17.CD19.1_3S (M1-M2: Cy3, RED) colocalizing with a CD19-specific antibody (N1-N2: APC, GREEN) on Raji cells. The aptamer demonstrates specificity and surface binding with no significant off-target interactions. Zoomed-in views (P1-P2) confirmed aptamer binding to the cell membrane. Scale bars = 7 and 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence. The aptamer and random mean fluorescence values correspond to the mean fluorescence observed in their respective histograms. Bar graphs represent mean ± standard deviation from three independent experiments with statistical significance indicated (****p < 0.0001). Data represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Fluorescence, Binding Assay, Confocal Microscopy, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization studies of CD19 Antibody and Bivalent CD19 Aptamers in Ramos and Raji Cells. (A–H) Time-course analysis of CD19 antibody and bivalent CD19 aptamer internalization in Ramos cells (A, C, E, and G: CD21-negative) and Raji cells (B, D, F, and H: CD21-positive). (A-B) demonstrate internalization of APC-CD19 antibody in Ramos (A) and Raji (B) cells. In Ramos cells (A), CD19 antibody shows efficient internalization over 48 hours, while in Raji cells (B), CD21 expression inhibits internalization. (C-D) show internalization of bivalent CD19 aptamer WB17/17.CD19.1_3S in Ramos (C) and Raji (D) cells. WB17/17.CD19.1_3S shows robust internalization in Ramos cells (C), but reduced uptake in Raji cells owing to CD21-mediated blocking. (E-F) illustrate internalization of bivalent CD19 aptamer WB15/17.CD19.1_3S in Ramos (E) and Raji (F) cells. The aptamer demonstrates high internalization efficiency in Ramos cells (E), but not CD21-positive Raji cells (F). Panels G and H: Internalization of bivalent CD19 aptamer WB15/15.CD19.1_3S in Ramos (G) and Raji (H) cells. Similar to other bivalent aptamers, WB15/15.CD19.1_3S internalizes efficiently in CD21-negative Ramos cells (G), but CD21 expression in Raji cells (H) significantly blocks its internalization. Data are expressed as the percentage of internalization calculated as . Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Blocking Assay, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Colocalization of CD19 and CD21 was confirmed by flow cytometry and confocal microscopy, whereas CD20 does not co-localize with CD21. (A) demonstrates flow cytometry histograms showing CD19 (A1: PE-CD19 mAb, Red) and CD21 (A2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (A3) Bi-parametric dot plot confirms colocalization of CD19 and CD21 on the same population of cells. (B) shows bar graph quantifying mean fluorescence intensity of CD19 and CD21, highlighting their robust expression on Raji cells. (C–E) Confocal microscopy images showing CD19 (C: PE-CD19, Red) colocalized with CD21 (D: APC-CD21, Light Blue) on the surface of Raji cells. Arrowheads indicate regions of colocalization (D). Panels F1–F3 illustrate flow cytometry analysis of bivalent CD19 aptamer WB17/17.CD19.1_3S (F1: Pink) binding to Raji cells and its colocalization with CD21 (F2: APC-CD21 mAb, Light Blue). (F3) Dot plot shows overlapping signals, confirming aptamer-CD21 interaction. (G) demonstrates the bar graph of mean fluorescence intensity of bivalent CD19 aptamer (WB17/17.CD19.1_3S) and CD21 antibody. (H–J) present confocal microscopy images showing WB17/17.CD19.1_3S (H: Red) colocalized with CD21 (I: Green) on Raji cells. Arrowheads highlight colocalized regions (J). (K1–K3) Flow cytometry analysis showing CD20 (K1: FITC-CD20 mAb, Green) and CD21 (K2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (K3) Dot plot shows no significant overlap between CD20 and CD21, indicating no colocalization. (L) Bar graph showing fluorescence intensity of CD20 and CD21. (M–O) Confocal microscopy images showing no colocalization between CD20 (M: Red) and CD21 (N: Green) on Raji cells (O). (P1–P3) Flow cytometry analysis of bivalent CD20 aptamer WB1/1.CD20.1_3S (P1: Blue) and CD21 (P2: APC-CD21 mAb, Light Blue). (P3) Dot plot confirms the absence of colocalization on the same population of cells. Panel Q: Bar graph of fluorescence intensity for CD20 aptamer and CD21. (R–T) Confocal microscopy images showing that WB1/1.CD20.1_3S (R: Red) and CD21 (S: Green) do not co-localize on Raji cells (T). Scale bars = 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Data represents mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence, Expressing, Binding Assay, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: The expression of CD19, CD20, and CD21, the binding activity of bivalent CD19 aptamers, and the internalization dynamics of CD19 antibody and bivalent aptamers in OCl-LY7 and HBL-1 (DLBCL) cells. Antibody staining for CD19, CD20, and CD21 expression. (C; G) demonstrate flow cytometry histograms showing the expression of CD19 (C2: Red), CD20 (C1: Green), and CD21 (C3: Light Blue) on OCI-Ly7 (C), whereas HBL-1 (G) cells express CD19 (G2: Red), CD20 (G1: Green), and CD21 (G3: Light Blue). Isotype controls (gray) confirm specific binding. (D; H) Bar graphs showing mean fluorescence intensities, confirming robust expression of CD19 and CD20, but the absence of CD21 expression in both cell lines. Binding assay with bivalent CD19 aptamers (A1–A4, E1–E4). Fluorescence intensity histograms of bivalent CD19 aptamers (WB17/17.CD19.1_3S, WB15/17.CD19.1_3S, and WB15/15.CD19.1_3S) compared to random DNA control in OCI-Ly7 (A1–A4) and HBL-1 (E1–E4) cells. (B; F) illustrate bar graphs of mean fluorescence intensities showing high binding specificity of bivalent aptamers compared to controls. Internalization of CD19 antibody with and without Proteinase K (I, K). Flow cytometry histograms (L1) and bar graphs (L2) showing internalization dynamics of CD19 antibody in OCI-Ly7 (I), whereas flow cytometry histograms (K1) and bar graphs (K2) show internalization dynamics of CD19 antibody in HBL-1 (K) cells. Internalization was analyzed at 0 hour and 24 hours in the presence and absence of Proteinase K. Data reveal a significant reduction in surface fluorescence intensity after Proteinase K treatment, confirming internalization. Internalization of bivalent CD19 aptamers under the same conditions (J, L). Fluorescence intensity histograms (J1-J3, L1-L3) and bar graphs (J4 and L4) show the internalization of bivalent CD19 aptamers (WB17/17.CD19.1_3S (J2, L2), WB15/17.CD19.1_3S (J1, L1), and WB15/15.CD19.1_3S (J3, L3)) in OCI-Ly7 (J) and HBL-1 (L) cells. Internalization is measured at 0 hour and 24 hours with and without Proteinase K, demonstrating the effective uptake of bivalent aptamers. Data are expressed as the percentage of internalization calculated as fluorescence intensity was calculated using the formula. Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Each bar represents mean ± standard deviation from three independent experiments. Data are presented as mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Binding Assay, Activity Assay, Staining, Flow Cytometry, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization Assay of CD19 Antibody and Bivalent CD19 Aptamer in Ramos and OCI-Ly7 Cells Visualized by Confocal Microscopy. Internalization of CD19 Antibody in Ramos Cells (A-D). Confocal images showing surface-bound APC-CD19 antibody (A1-A2: Red) at 0 hour with and without Hoechst nuclear staining (A2: Blue). (B1–B2) demonstrate the isotype control at 0 hour. Panels C1-C2 show the internalization of APC-CD19 after 24 hours (C1-C2: Red) with and without Hoechst nuclear staining (C2: Blue). (E) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in Ramos Cells (F-I). Initial binding of WB17/17.CD19.1_3S (F1-F2: Green) at 0 hours, as shown with Hoechst-stained nuclei (F2: Blue). (H1–H2) demonstrate internalization of WB17/17.CD19.1_3S at 24 hours with and without Hoechst nuclear staining (H2: Blue). (J) shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Internalization of CD19 Antibody in OCI-Ly7 Cells (K-N) Surface-bound APC-CD19 antibody at 0 hours (K1-K2). (M1–M2) illustrate reduced surface-bound fluorescence at 24 hours, consistent with internalization. After 24 hours, CD19 antibody is completely uptaken on OCl-Ly7 cells. (O) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in OCI-Ly7 Cells (P-S). Confocal images showing WB17/17.CD19.1_3S (P1: Green) binding at 0 hours with and without Hoechst nuclear staining (P2: Blue). (R1–R2) highlight internalized WB17/17.CD19.1_3S at 24 hours with Hoechst nuclear staining (R2). Panel T shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Scale Bars: 5 μm. Data represent mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Confocal Microscopy, Staining, Control, Fluorescence, Binding Assay, Standard Deviation

Journal: Experimental & molecular medicine

Article Title: Distribution and impact of p16 INK4A+ senescent cells in elderly tissues: a focus on senescent immune cell and epithelial dysfunction.

doi: 10.1038/s12276-024-01354-4

Figure Lengend Snippet: Fig. 4 The distribution of CDKN2A+ cells in normal lung and liver tissues from young and aged individuals in scRNA-seq. a Feature plots display the expression of CDKN2A in the public dataset of lung, SYN1041850. b Bar graphs depict the proportion of CDKN2A+ cells in each cell type of the lung tissue. c The multiplex IHC analysis shows each subtype of CD19+ B cells according to the p16INK4A expression. d Feature plots display the expression of CDKN2A in the public dataset of liver, GSE149614. e The quantification data of CDKN2A+ cells in each cell type of liver are shown. f The multiplex IHC analysis shows each subtype of CD68+ Kupffer cells according to the p16INK4A expression.

Article Snippet: The primary antibodies used for human samples were as follows: p16INK4A, predilution (805–4713, Roche, Basal, Switzerland); human Ki67, 1:3,000 (M7240, Dako, Glostrup, Denmark); H3K9me3, 1:500 (ab176916, Abcam, Cambridge, MA); CD3, 1:150 (ab135372, Abcam); CD19, 1:100 (ab227688, Abcam), CD68, 1:2,500 (NBP2-48923, Novus Biologicals, Centennial, CO); PAR1, 1:200 (ab233741, Abcam); PAR2, 1:100 (ab184673, Abcam); Granzyme A, 1:100 (ab209205, Abcam); Cleaved caspase-3, 1:400 (9661S, Cell Signaling Technology, Danvers, MA); IL8, 1:300 (27095-1-AP, Proteintech, Rosemont, IL) IFNα 1:200 (PA5-119649, Thermo Fisher Scientific, Waltham, MA); IFNβ 1:500 (PA5-102429, Thermo Fisher Scientific).

Techniques: Expressing, Multiplex Assay

Journal: Nature communications

Article Title: Engineering advanced logic and distributed computing in human CAR immune cells.

doi: 10.1038/s41467-021-21078-7

Figure Lengend Snippet: Fig. 5 Tunable 3-input multilogic in the single cell. a Design of orthogonal SUPRA CARs that control CD3ζ, CD28, and BTLA signaling domains inducibly and independently. Primary CD8+ T cells were engineered to express FOS zipCAR, SYN6 zipCAR and SYN1 zipCAR that contain CD3ζ domain, CD28, and BTLA signaling domain, respectively. In addition, α-Meso-SYN9 zipFv, α-Axl-SYN5 zipFv, and α-Her2-SYN2 zipFv lead to activation of CD3ζ, CD28, and BTLA, respectively. (Right) IFN-γ secretion was measured after co-culturing with Her2, Axl, and Meso expressing Nalm6 target cells with different zipFv combinations (n = 3, data are represented as the mean + SD, the statistical significance was determined by Student’s t-test). b Primary CD4+ T cells expressing FOS-CD3ζ, SYN6-CD28, and SYN1-BTLA were co-cultured with Her2, Axl, and CD19 expressing Nalm6 target cells. The 3D heatmap shows IFN-γ production from 3-input CD4+ T cells at varying concentrations of three different corresponding zipFvs (n = 2, data are represented as the mean).

Article Snippet: Also, fluorescent proteins and staining CD19 (anti-Human CD19 PE-Cy7 (1:200 dilution, Tonbo Bioscience, clone HIB19)) were used to further identify the target cells, or staining CD8 (BV421 Mouse AntiHuman CD8 (1:200 dilution, BD)) was used to exclude CAR-T cells This gate was applied to all other samples.

Techniques: Control, Activation Assay, Expressing, Cell Culture