... Protocols Fluorescence Titering Assay Fluorescence Titering Assay ...resulting in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent x 100)/(1.5 mL)...resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent)/(0.015 mL) = ...preparations carrying a fluorescence marker. Protocols...to titer lentiviral preparations that carry a fluorescence marker. Determining the titer of your lentiviral...uses microscopy to determine the percentage of fluorescent cells, these cells can still be used in downstream...media, replace with fresh media Day 4–5: Count fluorescent cells Equipment Class II, Type A2 Biological ...

...primary antibody against a target protein and a fluorescent secondary antibody....primary antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the... tips and pipettes Rocking platform Tweezers Fluorescent microscope 0.5–10 µL single channel pipette 2...500 µL PBS on a rocking platform. Dilute the fluorescently-labeled secondary antibody to the desired concentration...generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary antibody to the wells and ...cell labeling on a microscope with appropriate fluorescent filters. Tips and Troubleshooting The optimal...

...maintenance phase and do not allow cells to reach 100% confluence (80–90% is ideal). Pass and plate the cells the...from 2 x T-175 flasks. Cells should be at ~80% confluence. For each T-175 flask: Aspirate culture media...resuspend cells from the CS2. Cells should be at ~80% confluence. Aspirate culture media and rinse once with 60...Sample Data Figure 1: HEK293T cells at various confluencies. This AAV production protocol should be started...started with cells that are ~80% confluent (center panel). These cells were imaged with a 10X objective ...

...with fresh media Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment Equipment...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...efficiencies as can be seen here by the amount of green fluorescent protein expression (green in the right panels...

...the individual wells become confluent, expand into larger vessels. A confluent well of a 6-well dish can...can be expanded into a 10 cm dish. A confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc....

...the next day they will be approximately 50–60% confluent. For 293T cells this is about 2 × 10 6 cells in...Do not use the medium if the cells are overly confluent or the medium is acidified (i.e., the medium appears...cells have expanded but before they become over-confluent, trypsinize the cells and expand them to larger...

...cells from incubator. The cells should be 50-80% confluent and in DMEM that does not contain antibiotics....The target cells should be approximately 80-90% confluent. c. Dilute puromycin in the preferred culture ...: b. Target cells should be approximately 70% confluent. Change to fresh culture media containing 8 μg...

...transfection protocol Fluorescence Titering Assay for Lentivirus Quantify virus using fluorescence measurements...

...ROX dye), to normalize non-PCR–related fluorescence fluctuations and to minimize well-to-well variability...

...batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the ...

...different concentrations of DNA. In our hands fluorescent dye methods tend to report lower values than ...

... Colony Formation Titration Assay Lentivirus Fluorescence Titration Assay AAV Titration by qPCR Addgene...