... filter will maximize titer . DMEM high glucose, Corning 10-013-CV DMEM, low glucose (1 g/L) glucose, ...HCl, 150 mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ...Store at 4 °C. D1 + 0.1 M sorbitol (optional) : DMEM low glucose + 1% v/v HI-FBS + 0.1 M sorbitol In ...7.5% Sodium Bicarbonate, 7.5 mL 1 M HEPES to 750 mL DMEM + 1 g/L glucose. 1 mg/mL polyethylenimine (PEI) ...Wait ~2 min. Neutralize trypsin by adding 10 mL of DMEM Complete. Pipet back and forth vigorously multiple...from 2 x T-175 flasks. Adjust volume to 300 mL with DMEM complete media and mix. Seed all cells in 1 CS2....

...Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat inactivated FBS ...for the colony formation assay. Prepare a batch of DMEM complete containing 10 μg/mL polybrene by diluting... 10-fold serial dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene: Dilution...of Lentivirus or Previous Dilution (μL) Volume of DMEM Complete Containing 10 μg/mL Polybrene (μL) Volume...cells as follows: Dilute 7,000 cells into 9.45 mL of DMEM complete containing 10 μg/mL polybrene. Mix well...

...flask in 15 mL DMEM Complete. Wednesday: Plate 1×10 6 cells in a T75 flask in 15 mL DMEM Complete. Friday...container pH meter Stir plate Magnetic Stir Bar Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...encoding your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS and 4 ...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ...Friday: Plate 8×10 5 cells in a T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use...293T packaging cells at 3.8×10 6 cells per plate in DMEM Complete in 10 cm tissue culture plates. Incubate...15 min at RT. During the incubation, add 10 mL of DMEM Complete to a 15 mL conical and an appropriate amount...

...Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...e.g. puromycin, blasticidin) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ... that kills all of the cells. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL ...Prepare a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Note, this is just...well. Dilution Volume of Lentivirus (μL) Volume of DMEM complete + 10 µg/mL polybrene (µL) 0 0 500 1:5 300...Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting...

...fresh DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete...Pipet controller Hazardous waste container Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...filter Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ...Seed HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate...aspirate the media. Replace the media with 15 mL of DMEM complete. Incubate the cells 24–48 h before checking...

...Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate 55 mL heat inactivated...well tissue culture treated dish, VWR, 29442-042 DMEM, high glucose, pyruvate, Corning, 10-013-CV Heat-inactivated...bovine serum 5 mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing...Prepare the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge tubes with the...well). Leave one well untransduced (add 150 µL of DMEM complete instead of a viral dilution). Remember,...others. Gently add 1.5 mL of 50 U/mL Benzonase in DMEM complete to each well. Incubate at 37 °C for 30 ...µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge tube....

...container Inverted fluorescence microscope Reagents DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...21008-216 Lentivirus preparation Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ...cells as follows: Dilute 525,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot...to use. Prepare dilutions of the lentivirus into DMEM complete containing 10 μg/mL polybrene. Note, this...Dilution Volume of Lentivirus Stock (μL) Volume of DMEM complete (μL) Volume of 10mg/mL polybrene (μL) 1...

...cell counter Reagents Polyclonal stable cell pool DMEM high glucose, Corning 10-013-CV L-alanyl-L-glutamine...dish, Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine...alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add 55 mL of heat-inactivated FBS ... dish. Each 10 cm dish should be seeded in 10 mL DMEM complete, which will generate enough conditioned...

...passaged in DMEM + 10% FBS with penicillin/streptomycin, cells should be plated at this step in DMEM + 10% ...Invitrogen: #31985 Dulbecco’s Modified Eagle Medium (DMEM) Invitrogen: #11995 Fetal Bovine Serum (FBS) Invitrogen...incubator. The cells should be 50-80% confluent and in DMEM that does not contain antibiotics. h. Without touching...the transfection reagent. Replace with 5 mL fresh DMEM + 10% FBS + penicillin/streptomycin. Pipette the...

...Gel Electrophoresis Protocol with the following amendments: Notes: Gel purification is most efficient with...