4. Flush the system and the membrane stack with the running
Buffer A for at least 60 column volumes (see Note 27).
5. Load the sample from the syringe to the 10 mL sample loop (see
Note 28).
6. Perform the actual SXC runs comprising the following steps:
(a) Equilibration with at least 5 mL of buffer A (see Note 29).
(b) Sample application using the sample loop (see Note 30).
(c) Washing with at least 5 mL buffer A (see Note 31).
(d) Elution with 5–10 mL elution buffer B (see Notes 32–
34).
7. Fractionate the entire volumes for step 6b–d for off-line
analytics.
8. Clean the system using cleaning buffer and flush system afterward with water.
9. (a) Start next SXC purification or
(b) Fill system with 20% ethanol in water for storage.
10. Discard the used membranes and store the virus samples at
À80
C until performing the offline analytics (see Notes 35–39).
3.2.5 Quantification
of the Purified Virus
Particles by Flow
Cytometry
1. Prepare standard calibration range by diluting the virus stock
7 times with subsequent 1:2 dilutions in cell culture medium
(e.g., use 350 μL medium +350 μL preceding virus standard)
(see Notes 40 and 41).
2. Seed host cells at 1E+07 cells mL with 1 mL per well into the
24-well plate. For each standard and sample prepare three wells
of cells (see Note 42).
3. Infect the seeded cells directly with 100 μL of the virus suspension (100 μL/well for standards and samples).
4. Incubate at 37
C and 8% CO 2 for 16 h.
5. Discard the supernatant and wash all wells twice with
1 mL PBS.
6. Add 200 μL of trypsin–EDTA solution and allow incubation
for 3–5 min at 37
C.
7. Add 50 μL FCS.
8. Carefully resuspend the cells and transfer the content of each
well into a new well on the 96-well U-bottom plate.
9. Centrifuge the plate for 5 min at 500 Â g.
10. Discard the supernatant and resuspend cells in 100 μL PBS (see
Notes 43 and 44).
11. Measure the samples in the flow cytometer as follows.
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Keven Lothert et al.
Buffer A for at least 60 column volumes (see Note 27).
5. Load the sample from the syringe to the 10 mL sample loop (see
Note 28).
6. Perform the actual SXC runs comprising the following steps:
(a) Equilibration with at least 5 mL of buffer A (see Note 29).
(b) Sample application using the sample loop (see Note 30).
(c) Washing with at least 5 mL buffer A (see Note 31).
(d) Elution with 5–10 mL elution buffer B (see Notes 32–
34).
7. Fractionate the entire volumes for step 6b–d for off-line
analytics.
8. Clean the system using cleaning buffer and flush system afterward with water.
9. (a) Start next SXC purification or
(b) Fill system with 20% ethanol in water for storage.
10. Discard the used membranes and store the virus samples at
À80
C until performing the offline analytics (see Notes 35–39).
3.2.5 Quantification
of the Purified Virus
Particles by Flow
Cytometry
1. Prepare standard calibration range by diluting the virus stock
7 times with subsequent 1:2 dilutions in cell culture medium
(e.g., use 350 μL medium +350 μL preceding virus standard)
(see Notes 40 and 41).
2. Seed host cells at 1E+07 cells mL with 1 mL per well into the
24-well plate. For each standard and sample prepare three wells
of cells (see Note 42).
3. Infect the seeded cells directly with 100 μL of the virus suspension (100 μL/well for standards and samples).
4. Incubate at 37
C and 8% CO 2 for 16 h.
5. Discard the supernatant and wash all wells twice with
1 mL PBS.
6. Add 200 μL of trypsin–EDTA solution and allow incubation
for 3–5 min at 37
C.
7. Add 50 μL FCS.
8. Carefully resuspend the cells and transfer the content of each
well into a new well on the 96-well U-bottom plate.
9. Centrifuge the plate for 5 min at 500 Â g.
10. Discard the supernatant and resuspend cells in 100 μL PBS (see
Notes 43 and 44).
11. Measure the samples in the flow cytometer as follows.
236
Keven Lothert et al.