1, Grow Sf9 cells in 2 l Sf-900 II medium in Erlenmeyer flasks at 27 oC (orbital shaker at 110 rpm)
2, Infect cells (1~2 10^6 cells per ml density) using an MOI of 0.1 PFU per cell.
3, Harvest cells 5 days after infection (when the cell viability reached 50%, assayed using 0.4% trypan blue exclusion dye).
4, Spin culture at 1500 g for 20 min.
5, Collect supernantant and store at 4 oC in the dark.
6, Filter through a 0.65 µM filter.
7, Concentrate solution by cross-flow filtration (diafiltration + tangential cross-flow filtration) using an AKTA system (100 kDa  Sartorius membrane, 35 ml/min retentate flow rate and TMP of 1.2 bars)
8, Purify virus by Sartobind D MA 75 units (Sartorius Stedim Biotech) with 75 cm2 effective membrane area (equivalent to approximately 2 ml of membrane volume) containing DEAE, or a 10 ml Capto DEAE column

FPLC purification:
9, Equilibrate column/membrane with Dulbecco’s phosphate-buffered saline (D-PBS).
10, Load 100 ml sample
11, Flow rates:  5 ml/min for resin and 10 ml/min for membrane.
12, Elute virus with 600 mM NaCl in D-PBS.
13,  Pool fractions. Analyse fractions by SDS–PAGE and qPCR
14,  Desalt with a HiPrep 26/10 desalting column
15,  Filter purified virus with Acrodisc Syringe Filters with Supor PES Membrane (Pall) or Sartorius Minisart NML (Sartorius Stedim Biotech).
16, Store virus at 4 oC in the dark.

1, Lyse cells with cell lysis buffer containing 0.1% Triton X-100.
2, Add Benzonase (15 U/mL) and incubate for 30 min at 37 oC.
3, Spin and pass supernatant through a Sartobran 0.45-um filter.
4, Optional: concentrate supernatant with 300 MWCO polysulfone hollow-fiber cartridges (GE).
5, Adjust supernatant to pH 8.0, or buffer exchange  by diafiltration with Vivaspin 20 with 100 kDa cut-off.
6, Load 50 ml supernatant to Sartobind Q MA75 and Sartobind anion direct (2.5 mL) pre-equilibrated with loading buffer (10 mM Tris–HCl pH 8.0), flow rate 3 ml/min.
7, Wash column with 50 ml of washing buffer to wash contaminated proteins (250 mM NaCl, 10 mM Tris–HCl pH 8.0) at 3 ml/min.
8, Elute adenovirus with 20 ml of elution buffer (0.65 M NaCl, 10 mM Tris–HCl pH 8.0) at 3 ml/min.
9, Regenerate  column with 20 ml regeneration buffer  (1 M NaCl, 10 mM Tris–HCl pH 8.0) at 3 ml/min.
10, Concentrate fractions containing adenovirus using polysulfone hollow-fiber cartridge with 300 kDa cut-off  (GE Healthcare).
11, Filter concentrated adenovirus through a sterile Filtropur S 0.2 um filter (Sarstedt)
12, Store adenovirus ay 4 oC.
13, Analyse adenovirus purity by size exclusion chromatography using a HiPrep 16/60 Sephacryl S-300 column (1.6 X 60 cm) or SDS-PAGE.

Sartobind anion direct is better than Sartobind Q MA75 for purification of adenoviruses
Sartobind Q MA75 requires recirculation for adenovirus capturing.

1.     Seed cells into the tissue culture flask at approximately 4 x 10^4 cells per cm2.
2.     Feed cells with recommended media (for example, DMEM, high glucose with 4 mM glutamine,  and 10% Fetal Calf Serum,  with antibiotics and supplements if required).
3.     Culture cells until 70 to 80% confluency is achieved.
4.     Transfect the cultures with packaging plasmid mix and expression construct. Collect the viral supernatant from the transfected cells 2 to 3 days after transfection.
5, Pool the viral supernatant into 50 mL centrifuge tubes or capped vessels.
6.     Centrifuge at 3000 rpm for 5 minutes. Collect the supernatant into clean 50 mL polypropylene centrifuge tubes; discard the pellet(s). The solution should be free of observable debris. If any traces of debris are observed, centrifuge the viral supernatant again.
7, Add Benzonase nuclease (1 μL for each 10 mL of viral supernatant), or the equivalent DNase (100 Kunitz units for each 10 mL of viral supernatant). Cap container and gently invert the solution to mix thoroughly. Incubate at 37 °C for 30 minutes
8, Add 10X binding buffer to viral supernatant
9, Purify lentivirus by anion-exchange chromatography

1, Harvest of lentivirus
2, Benzonase treatment 30 minutes at 37 °C
3, Purification with Fast-Trap Lentivirus Purification & Concentration Kit (Millipore #FTLV00003):
4, Clarify using provided 0.45 μm HV Steriflip® filter unit by vacuum filtration
5, Purify virus using Fast-Trap purification device & buffers
6, Concentrate/buffer exchange of eluted lentivirus with supplied 100 kDa Amicon® Ultra device.

Binding Capacity of Mustang Q membrane column: 4.9 x 10^13 VP/mL of membrane.

1, Harvest infected cells by pelleting and resuspending the pellet in PBS.

2, Lyse host cells by three freeze-thaw cycles and separate the cell debris by spinning down at 20,000 x g for 60 minutes at 4 oC.

3, Filter lysate through a 25 mm Acrodisc syringe filter with 1.2 μm Supor®membrane followed by a 25 mm Acrodisc syringe filter with 0.2 μm Supor membrane.
This two-step filtration process may require more then just a single Acrodisc filter depending on the effectiveness of the pelleting procedure.

4, Incubate filter lysate at room temperature for 30 minutes with either 100 units of Benzonase or 100U DNAase/RNAse cocktail per mL of cell lysate.

5, Filter with 0.2 μm filtration (25 mm Acrodisc PF syringe filter with Supor membrane, PN 4187) to ensure that the lysate is free of particulate matter.

6, Adjust lysate to a final concentration of 0.3 M NaCl.

7, Precondition Mustang Q membrane column (PN MSTG25Q6) with 0.3 M NaCl in 25 mM HEPES, pH 7.4.

8, Load lysate (with 0.2 M or 0.3 M NaCl) to a 0.18 mL bed volume (BV) Mustang Q column (ÄKTA Explorer* 10).

9, Eluted viral particles at 20 column volumes (CV)/min (3.5 mL/min) using buffer A (0.2M NaCl in 20mM HEPES pH 7.5, 1mM MgCl2) and buffer B (1M NaCl in 20mM HEPES pH 7.5, 1mM MgCl2).
   The gradient is 0 to 40% B in 0.5 CV, then held at 40% B for 20 CV and a final elution gradient of 40 to 100% B in 10 CV.
   Collect 0.5 mL fractions.
    Viral peak will be at 30 mS/cm in the 0 to 40% B gradient.

10, Combine fractions containing adenovirus, add sucrose to a final concentration of 8%.

11, Concentrate and desalte virus prep on a Microsep centrifugal device with 300K Omega membrane (PN OD300C41 or OD300C46).

Advantage:

1, Purify 1^10  viral particle in  30 minutes.
2, Adenovirus purified with membrane has lower cytotoxicity in primary neurons than CsCl-purified virus.

Disadvantage:
    Higher protein content than CsCl-purified virus