4. Microplate spectrophotometer should have dual wavelength
capacities to be able to read OD 600 and OD 570 in the same
reading.
5. Unless otherwise specified, all solutions should be prepared
using ultrapure, endotoxin-free water, and analytical grade
reagents. Prepare and store all reagents at RT unless indicated
otherwise. Follow all waste disposal regulations when disposing
of chemical and biological waste materials.
6. Spent culture media (CFP) must be sterile-filtered prior to
removal from a BSL-3 laboratory. If CFP is not processed
immediately, 0.02% sodium azide, or another bacteriostatic
chemical, should be added to prevent contamination.
7. Multiple sizes of stirred ultrafiltration cells are available. Select
the size that is appropriate for your sample volume.
8. Vessel fittings for the inlet should be compatible with tubing
from the nitrogen source; a ball valve and quick-disconnect
nipple are recommended. Fittings for the vessel outlet must
be compatible with the stirred ultrafiltration cell inlet tubing.
9. Multiple sizes are available. Select the appropriate diameter for
the stirred ultrafiltration cell.
10. Multiple sizes are available. Select the appropriate size for the
anticipated final concentrate volume.
11. Any protein quantitation method may be used as long as it is
compatible with the buffers used. Be sure to use protein standards made in the same buffer as your sample.
12. Avoid the use of protein-rich media supplements like OADC
when growing cells for CFP harvest and MEV purification.
13. Other size-exclusion chromatography resins can be used.
However, traditional resins lack the binding properties and
resulting dual functionality of Capto™ Core 700 and
non-MEV proteins will eventually elute. The vesicle enrichment described in this protocol is specific to this multimodal
resin.
14. Alternative ultrasensitive protein quantitation method may be
used as long as it is compatible with the buffers used. Be sure to
use protein standards made in the same buffer as your sample.
15. Alternative vesicle quantification and sizing techniques can be
applied; these include but are not limited to: Dynamic Light
Scattering (DLS) and Tunable Resistive Pulse Sensing (TRPS).
16. It is important to keep the buffer cool (in a refrigerator or on
ice) after the addition of the protease inhibitor tablet, DNase,
and RNase. Adjust stocks and protease inhibitor quantities
accordingly depending on the volume of breaking buffer
needed.
100
Megan Lucas et al.
capacities to be able to read OD 600 and OD 570 in the same
reading.
5. Unless otherwise specified, all solutions should be prepared
using ultrapure, endotoxin-free water, and analytical grade
reagents. Prepare and store all reagents at RT unless indicated
otherwise. Follow all waste disposal regulations when disposing
of chemical and biological waste materials.
6. Spent culture media (CFP) must be sterile-filtered prior to
removal from a BSL-3 laboratory. If CFP is not processed
immediately, 0.02% sodium azide, or another bacteriostatic
chemical, should be added to prevent contamination.
7. Multiple sizes of stirred ultrafiltration cells are available. Select
the size that is appropriate for your sample volume.
8. Vessel fittings for the inlet should be compatible with tubing
from the nitrogen source; a ball valve and quick-disconnect
nipple are recommended. Fittings for the vessel outlet must
be compatible with the stirred ultrafiltration cell inlet tubing.
9. Multiple sizes are available. Select the appropriate diameter for
the stirred ultrafiltration cell.
10. Multiple sizes are available. Select the appropriate size for the
anticipated final concentrate volume.
11. Any protein quantitation method may be used as long as it is
compatible with the buffers used. Be sure to use protein standards made in the same buffer as your sample.
12. Avoid the use of protein-rich media supplements like OADC
when growing cells for CFP harvest and MEV purification.
13. Other size-exclusion chromatography resins can be used.
However, traditional resins lack the binding properties and
resulting dual functionality of Capto™ Core 700 and
non-MEV proteins will eventually elute. The vesicle enrichment described in this protocol is specific to this multimodal
resin.
14. Alternative ultrasensitive protein quantitation method may be
used as long as it is compatible with the buffers used. Be sure to
use protein standards made in the same buffer as your sample.
15. Alternative vesicle quantification and sizing techniques can be
applied; these include but are not limited to: Dynamic Light
Scattering (DLS) and Tunable Resistive Pulse Sensing (TRPS).
16. It is important to keep the buffer cool (in a refrigerator or on
ice) after the addition of the protease inhibitor tablet, DNase,
and RNase. Adjust stocks and protease inhibitor quantities
accordingly depending on the volume of breaking buffer
needed.
100
Megan Lucas et al.