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Base medium (e.g., DMEM, 1640, or MyCo5A) supplemented with 10–20% fetal bovine serum (FBS) and 1% penicillin-streptomycin dual antibiotics.
Breast cancer cell lines MCF-7 and MDA-MB-231.
LentiCRISPRv2-CMV-ZsGreen-TEAD4-KO plasmid carrying green fluorescent protein and designed for TEAD4 knockout.
1)Pick single bacterial colonies from agar plates using a sterile pipette tip.
2)Inoculate the colonies into liquid LB medium.
3)Incubate overnight at 37 °C in a shaker for ~12 hours.
Standard LB Medium Recipe (per 1 liter)
Tryptone (peptone): 10 g
Yeast extract: 5 g
NaCl (sodium chloride): 10 g
Distilled water: to 1,000 mL
Preparation Steps
Weigh and dissolve: Add 10 g tryptone, 5 g yeast extract, and 10 g NaCl into ~800 mL distilled water in a beaker. Stir until fully dissolved.
Adjust volume: Add distilled water to bring the total volume up to 1,000 mL.
Adjust pH (optional but common): Set pH to 7.0 using NaOH or HCl if needed.
Sterilize: Dispense into bottles or flasks (fill only ~2/3 full).
Autoclave at 121 °C for 15–20 min.
Cool and store: Allow to cool to room temperature. Store at room temperature or 4 °C depending on usage frequency.
Variants LB Agar: Same recipe + 15 g agar (for plates).
( Tips: Always label the bottle with medium type, date, and your initials. If antibiotics (e.g., ampicillin, kanamycin) are required, add them after autoclaving and cooling to ~50 °C to avoid heat degradation.)
1)Add 500μL of equilibrium solution BL to the adsorption column CP3.Centrifuge at 12000 rpm for 1 minute. Repeat this process twice. Each time, discard the waste liquid in the collection tube.
2)Take 5ml of the overnight culture solution and add it to 5ml of glycerol, use a centrifuge at 12000 rpm per minute. Use a centrifuge, centrifuge at 12000 rpm for 1 min, centrifuge a total of 5 times. Each time, pour off the waste liquid, and then put the adsorption column back into the collection tube. (Try to absorb and remove the supernatant as much as possible)
3)Add 250 μL of P₁ (containing RNase) to the centrifuge tube with the bacterial pellet left, and use a vortex shaker to completely resuspend the bacterial pellet. (Scrape on the multi - well plate before use)
4)Add 250 μL of P₂ solution to the centrifuge tube, gently invert up and down 6 - 8 times to make the solution clear and thick.
5)Add 350 μL of P₃ solution to the centrifuge tube again, gently invert up and down 6 - 8 times until white flocculent precipitation appears.
6)Use a centrifuge, centrifuge at 12000 rpm for 10 min.
7)Use a pipette to transfer the clear liquid collected in the previous step to the adsorption column CP3 (Do not absorb the precipitate!)
8)Use the centrifuge again, centrifuge at 12000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorption column CP3 into a new collection tube.
9)Use the centrifuge to idle for 2 min at 12000 rpm.
10)Add another 50 μL of hot water for elution, let it stand for 2 min.
11)Use the centrifuge, centrifuge at 12000 rpm for 2 min.
12)Take 1 μL of the solution in the collection tube to measure the concentration.
1)Prior to thawing, turn on the biosafety cabinet and preheat a water bath to 37 °C. Retrieve the cryovial containing the cell line from liquid nitrogen storage and promptly place it in the 37 °C water bath. Once the contents have just fully thawed, immediately transfer the vial to the biosafety cabinet.
2)Inside the cabinet, transfer the thawed cell suspension into a 15 mL centrifuge tube containing 4 mL of complete medium. Mix gently by pipetting, centrifuge at 1,000 rpm for 5 minutes, and remove the supernatant.
3)Resuspend the cell pellet in 8 mL of fresh complete medium and transfer the suspension into a 9 cm culture dish. Gently shake the dish to evenly distribute the cells. Incubate the cells at 37 °C with 5% CO₂ until they adhere to the surface. These cells can then be used for subsequent experiments.
1)Before passaging, observe the cells under an inverted microscope to confirm that the confluence is approximately 80% and the cells are in good condition.
2)Using a 9 cm dish as an example: Transfer the dish to the biosafety cabinet and aspirate the culture medium. Rinse the cells with 3–4 mL PBS, aspirate the PBS, and add 1 mL of 0.125% trypsin. Gently rock the dish to ensure even coverage. Aspirate the trypsin and incubate the dish in the incubator to allow enzymatic digestion.
3)When the cells become round and detach (as seen under the microscope), add 2–4 mL of complete medium to terminate digestion. Gently pipette to detach and resuspend the cells, discard a portion of the suspension, retain an appropriate number of cells, and supplement with medium up to 8 mL. Return the dish to the incubator for continued culture.
1)Prepare cryopreservation solution with 90% FBS and 10% DMSO.
2)After trypsinization, resuspend the cells in 4 mL of complete medium, transfer to a 15 mL centrifuge tube, and centrifuge at 1,000 rpm for 5 minutes.
3)Remove the supernatant and add an appropriate volume of cryopreservation solution based on pellet size. Mix well, transfer to cryovials, and label with cell name, source, and freezing date. Place cryovials in a controlled-rate freezing container and store at −80 °C overnight before transferring to liquid nitrogen for long-term storage. Proper records should be maintained for both cell recovery and cryopreservation.
1)Seed cells in a 6-well plate and allow them to grow to approximately 80% confluence with healthy morphology.
2)Prepare two tubes with 100 μL serum-free medium each. To one tube, add 1 μg, 2 μg, or 4 μg of the target plasmid. To the other tube, add 4 μL of Lipo3000 transfection reagent. Mix both tubes thoroughly. Slowly add the plasmid solution into the transfection reagent tube, incubate at room temperature for 10–15 minutes, and then add the mixture to the culture wells. Gently swirl to mix.
3)After 6–8 hours of incubation, aspirate the transfection medium and replace it with complete medium. Continue culturing for an additional 12 hours.
4)Transfected cells can be harvested for downstream experimental procedures.
1)Seed MCF-7 cells at 4,000 cells/well and MDA-MB-231 cells at 3,000 cells/well into a 96-well plate. Each well should be supplemented to 100 μL with complete medium. Each experimental condition is performed in triplicate.
2)Once the cells adhere, add 10 μL of CCK-8 reagent to each well. Incubate at 37 °C protected from light for 1 hour. Measure absorbance at 450 nm wavelength. This time point is designated as 0 hours.
3)Repeat the assay at 24 and 48 hours. Analyze the effect of TEAD4 knockout on cell proliferation by comparing absorbance values over time.
1)Seed cells in a 12-well plate and allow them to reach approximately 90% confluence with healthy morphology. Use a sterile 200 μL pipette tip with a flat end to create a scratch by dragging it straight across the cell monolayer with a ruler for guidance to maintain linearity.
2)After scratching, aspirate the medium, wash once with PBS, then add 1 mL PBS per well. Capture images under an inverted microscope to record the initial wound width. Aspirate the PBS and replace it with fresh culture medium. Continue incubation.
3)After 24 hours, take images again to assess wound closure. Evaluate the effect of TEAD4 knockout on cell migration based on wound healing progress.
1)In the upper chamber of a Transwell insert placed in a 24-well plate, seed 2–5 × 10⁶ cells per well in 400 μL serum-free medium. Add 400 μL of complete medium to the lower chamber.
2)After 24 hours of incubation, remove the insert and aspirate the medium.
3)Place the insert in a new 24-well plate containing crystal violet stain. Ensure that the bottom side of the insert is submerged in stain. Incubate for 30 minutes at room temperature, protected from light.
4)Remove the dye and wash the insert three times with distilled water. Gently wipe off non-migrated cells from the upper side of the membrane with a cotton swab. Capture images under a microscope and quantify cell migration rates.