...media and cells. Centrifuge at 3900 rpm for 20 min at 4 °C to pellet the cells. Keep the cell pellet on ice...Seed cells in CS2 Day 2: Seed cells in CS5 Day 3 (am): Transfect cells Day 7 (am): Harvest cells Equipment...resuspend each pellet completely. Combine resuspended pellets and keep on ice. Process the cell pellet from above... HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells. Pass the cells twice a week...multiple times to obtain a single cell suspension (no clumps of cells). Pool cells from 2 x T-175 flasks. Adjust...obtain a single cell suspension (no clumps). Count cells using a hemocytometer or cell counter. Seed 350...Harvest cells and media by tapping the sides of the CS5. Cells should detach easily. Transfer cells and media...

...control well). Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish.... Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well of the 6-well dish. This brings ... the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the...single viral dilution to each well (each well gets one dilution, so a 6-well plate will hold 5 dilutions...media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute...stable cell pool. Observe the dish every day to ensure that the cells in the untransduced well (0 µL lentivirus...conditions for the surviving cells. Even in the absence of cell death, the cell media should be changed every...

...0.5 cells/well. Seeding an average of 0.5 cells/well ensures that some wells receive a single cell, while...Seed individual cells in a 96-well plate Day 2–14: Monitor cells for growth and expand cells Day 14–30: Analyze...homogenized cell solution. Homogenized cell solution concentration: 4 × 10 5 cells/mL To seed one 96-well plate...of a 5 cell/mL solution. Calculate the total cells needed: Total cells needed: 10 mL × 5 cells/mL = 50...any well receives more than one cell. Pro-Tip In each 96-well plate, seed one of the corner wells with...within each well, especially in the corner of the well as cells tend to settle there. Once the cells have expanded...generate a monoclonal cell line from a polyclonal pool of stable cells. Transducing cells with lentivirus results...

...1,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 7,000 cells into...polybrene. Mix well by pipetting or inverting. Aliquot 1.35 mL of the cell suspension into each well of the 6... of selection, no cells have survived in the untransduced well, while lawns of cells are present in the...protocol was developed for A549 cells but can be adapted to a variety of target cell lines and selection markers...the cells cannot be used for later experiments. Workflow Timeline Day 0: Seed and transduce cells Day ...health of the target cell line is critical for obtaining accurate titers. Check the cells for mycoplasma (...regularly Do not over or under-grow your cells. Thaw a new vial of cells after 20–30 passages. Do not add penicillin...

... viral dilution to a well of a 6-well plate (1 dilution per well). Leave one well untransduced (add 150...1: Seed and transduce cells Day 4: Treat cells with Benzonase and harvest cells Day 4+: ddPCR and analysis...below). Seed 300,000 cells/well in 1350 µL media and 11.1 µg/mL Polybrene into the wells containing the virus... for 10 wells with 3,000,000 cells in 13.5 mL of media and 11.1 µg/mL Polybrene. Mix the cell suspension...suspension well before seeding. Mix each well with a 1 mL pipette 5–10 times. The final volume in the well is ...the 6-well plates. Be sure to use a new aspirating pipette for each well as to not contaminate cell conditions...pipette for each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL ...

...Procedure Seed 75,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute ...,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension...suspension into each well of the 6-well dish. Incubate the cells overnight. If using freshly collected virus...dilutions well Gently aspirate media from the cells. Add 1.5 mL of a viral dilution to each well (each well... well gets one dilution with one well remaining as the untransduced control). Count the cells in the remaining...fluorescent-positive cells in each well. When calculating titer, only consider wells with less than 40% ...: If 150,000 cells were transduced in the 1:100 well, resulting in 25% fluorescent cells, then the titer...

...transfected cells. k. Incubate cells at 37°C, 5% CO 2 for 24 hours. Day 4: l. Harvest media from cells and transfer... infect the target cells. Addition of puromycin will allow you to select for cells that stably express... 7×105 HEK-293T cells in 5 mL of media in a 6 cm tissue culture plate. Incubate cells at 37°C, 5% CO 2...Although cells should regularly be passaged in DMEM + 10% FBS with penicillin/streptomycin, cells should... temperature. g. Retrieve HEK-293T cells from incubator. The cells should be 50-80% confluent and in DMEM...you do not want to dislodge the cells from the plate. i. Incubate cells at 37°C, 5% CO 2 for 12-15 hours...antibiotics to the cells and incubate at 37°C, 5% CO 2 for 24 hours. Day 5: n. Harvest media from cells and pool...

...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...10 µL of the cell suspension/trypan blue mix into a cell counting chamber. Load the cell counting chamber...HEK293 cells and media to 50 mL conical tubes. Centrifuge for 15 min at 3100 x g to pellet the cells. Filter...interest in cell culture. This protocol describes how to transfect suspension HEK293 cells with recombinant...Section 1: Seeding cells The day prior to transfecting, seed a 108 mL culture of HEK293 cells at a density ...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability...transfer 0.5 mL of HEK293 cell suspension into a clean microcentrifuge tube. Pro-Tip Cells settle quickly and...

... the pellet. Air-dry the pellet for 5-10 minutes. Critical It is important to not let the pellet get too... or cells without a kit. The general steps of this protocol include homogenization/lysis of cells or tissues...lyse tissues or cells in Solution D. For tissues: use 1 mL of Solution D per 100 mg of cells. For cultured...cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s) to sit at room temperature for 5...effectively lyse open the cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL...gel-like white pellet of total RNA in the bottom of the tube. Wash the RNA by resuspending the pellet in 0.5–...

...first three wells of each row, using a different standard solution for each row of a 96-well ELISA microplate...remove the plate seal from the 96-well plate and set aside. Aspirate the wells and use a multichannel pipette...Solution directly to the wells to stop the reaction. Measure the absorbance of each well at 450 nm on the spectrophotometer...options available. Caution Use the Stop Solution in a well ventilated area and avoid breathing in the vapors...