Journal: STAR Protocols

Article Title: Inducing T cell dysfunction by chronic stimulation of CAR-engineered T cells targeting cancer cells in suspension cultures

doi: 10.1016/j.xpro.2022.101954

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Article Snippet: PE-labeled Monoclonal Anti-FMC63 scFv Antibody, Mouse IgG1 (Y45) , Acro Biosystems , Cat# FM3-HPY53; RRID: AB_2921284.

Techniques: Recombinant, Reverse Transcription, Software, Pore Size, Flow Cytometry, Transfection

Journal: PLoS ONE

Article Title: Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

doi: 10.1371/journal.pone.0254739

Figure Lengend Snippet: HEK293T cells were seeded into a 96-well plate on day 1. The next day, the cells were transduced with diluted lentiviral vector and the transduction enhancer polybrene. 24 h post-infection the transduction mix was removed and replaced by a mixture of staining reagents containing FabFluor-488 labeled anti-FMC63 scFv antibody, Cytotox Red, and Opti-Green background suppressor. Dead cells were stained by Cytotox Red. Infected cells expressing the anti-CD19 CAR were stained by the FabFluor-488 labeled anti-FMC63 scFv antibody. Viable non-infected cells remained unstained. Quantification was performed by real-time fluorescence imaging from days 3-5.

Article Snippet: The anti-FMC63 scFv mouse IgG1 antibody (AcroBiosystems) binding the CD19-CAR construct was used at a final assay dilution of 1:200 and the final concentration of FabFluor-488 was 2.5 μg/mL.

Techniques: Transduction, Plasmid Preparation, Infection, Staining, Labeling, Expressing, Fluorescence, Imaging

Journal: PLoS ONE

Article Title: Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

doi: 10.1371/journal.pone.0254739

Figure Lengend Snippet: (A) Phase contrast image of HEK293T cells (left), merged with phase contrast area analysis mask in yellow (right). (B) HEK293T cells infected with lentiviral vector at a 1:625 dilution. The expressed CAR-construct was stained with an anti-FMC63 scFv antibody labeled with FabFluor-488. Phase contrast image merged with green fluorescence channel (left), background corrected green fluorescence channel (middle), and phase contrast image merged with green fluorescence analysis mask in magenta (right). (C) Live/dead staining positive control cells treated with 0.005% Triton X-100 and stained with Cytotox Red. Phase contrast image merged with red fluorescence channel (left), background corrected red fluorescence channel (middle), and phase contrast image merged with red fluorescence analysis mask in blue (right). All images were taken at 10x magnification.

Article Snippet: The anti-FMC63 scFv mouse IgG1 antibody (AcroBiosystems) binding the CD19-CAR construct was used at a final assay dilution of 1:200 and the final concentration of FabFluor-488 was 2.5 μg/mL.

Techniques: Infection, Plasmid Preparation, Construct, Staining, Labeling, Fluorescence, Positive Control

Journal: PLoS ONE

Article Title: Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

doi: 10.1371/journal.pone.0254739

Figure Lengend Snippet: (A) The FabFluor-488 positive area was normalized to the phase contrast area over time after transduction. The IgG1 isotype negative control and the lentiviral vector (LV) negative control (matrix) showed no FabFluor488 signal. The anti-transferrin-receptor IgG1 positive control antibody confirmed successful FabFluor488 labeling of the test antibodies. Matrix control overlaps with the negative control. Data represent the mean ± standard deviation of three technical replicates. (B) Normalized FabFluor488 peak value vs. time at which this value is reached. The datapoint in the upper left with the highest normalized positive area represents the 1:2 LV dilution. The other datapoints represent two-fold serially diluted LV up to a dilution of 1:1024 with the lowest normalized positive area and the longest time after which the peak value is reached. Data represent the mean ± standard deviation of three biological replicates.

Article Snippet: The anti-FMC63 scFv mouse IgG1 antibody (AcroBiosystems) binding the CD19-CAR construct was used at a final assay dilution of 1:200 and the final concentration of FabFluor-488 was 2.5 μg/mL.

Techniques: Transduction, Negative Control, Plasmid Preparation, Positive Control, Labeling, Control, Standard Deviation