6. The plastic bottles used for the centrifugation of the leptospiral
cultures should be dedicated to such task and should not be
used with any other bacteria and/or detergent to prevent any
endotoxin contamination.
7. It is recommended to check before doing the purification that
the 50 mL plastic tubes used are appropriate for hot phenol
extraction and that there is no degradation of the plastic during
the heating process.
8. The expected yield of the LPS extraction depends mainly on
the quality of the pellet resuspension in hot water. This step
must not be underestimated and should last at least 3–5 min
depending on the size of the pellet.
9. In order to prepare the 90% phenol solution required for the
extraction, the pure solid phenol must be heated to temperature above 68
C and then mixed to endotoxin-free water. The
solution must be kept in a water bath above 68
C in order to
ensure the proper water/phenol miscibility. Otherwise, the
extraction will not be performed properly.
10. It is quite frequent that the two phases are inverted during the
centrifugation and that the phenol phase is above the aqueous
phase.
11. It is recommended to conserve all the phases obtained during
the extraction to control the potential presence of LPS even in
the aqueous phase.
12. If the aqueous phase is not completely transparent, and contaminated by debris, it is advised to repeat one more time the
extraction step. Indicative volumes for second extraction:
10 mL of water and 10 mL of 90% phenol.
13. The dialysis tubes must be rehydrated at least 2 h prior to usage
and should also be pre-warmed up to avoid breaking when
thrown in hot water. They should also be washed with
endotoxin-free water.
14. The limulus amebocyte lysate (LAL) test, which traditionally
allows a precise quantification of endotoxins, is not useful when
it comes to the quantification of the leptospiral LPS. Indeed,
Leptospira LPS barely react with the lysate and usually exhibit
negative or results 1000-fold lower than with the same amount
of E. coli LPS [4].
15. In order to determine the number of active molecules present
in the LPS sample, one possibility is to stimulate HEK293TNFκB reporter cells transfected with the mTLR4 receptor.
Indeed as described earlier, mTLR4 recognizes the lipid A
part of the leptospiral LPS, and cells respond proportionally
to the number of molecules present in the purification. This
Purification of LPS from Leptospira
63
cultures should be dedicated to such task and should not be
used with any other bacteria and/or detergent to prevent any
endotoxin contamination.
7. It is recommended to check before doing the purification that
the 50 mL plastic tubes used are appropriate for hot phenol
extraction and that there is no degradation of the plastic during
the heating process.
8. The expected yield of the LPS extraction depends mainly on
the quality of the pellet resuspension in hot water. This step
must not be underestimated and should last at least 3–5 min
depending on the size of the pellet.
9. In order to prepare the 90% phenol solution required for the
extraction, the pure solid phenol must be heated to temperature above 68
C and then mixed to endotoxin-free water. The
solution must be kept in a water bath above 68
C in order to
ensure the proper water/phenol miscibility. Otherwise, the
extraction will not be performed properly.
10. It is quite frequent that the two phases are inverted during the
centrifugation and that the phenol phase is above the aqueous
phase.
11. It is recommended to conserve all the phases obtained during
the extraction to control the potential presence of LPS even in
the aqueous phase.
12. If the aqueous phase is not completely transparent, and contaminated by debris, it is advised to repeat one more time the
extraction step. Indicative volumes for second extraction:
10 mL of water and 10 mL of 90% phenol.
13. The dialysis tubes must be rehydrated at least 2 h prior to usage
and should also be pre-warmed up to avoid breaking when
thrown in hot water. They should also be washed with
endotoxin-free water.
14. The limulus amebocyte lysate (LAL) test, which traditionally
allows a precise quantification of endotoxins, is not useful when
it comes to the quantification of the leptospiral LPS. Indeed,
Leptospira LPS barely react with the lysate and usually exhibit
negative or results 1000-fold lower than with the same amount
of E. coli LPS [4].
15. In order to determine the number of active molecules present
in the LPS sample, one possibility is to stimulate HEK293TNFκB reporter cells transfected with the mTLR4 receptor.
Indeed as described earlier, mTLR4 recognizes the lipid A
part of the leptospiral LPS, and cells respond proportionally
to the number of molecules present in the purification. This
Purification of LPS from Leptospira
63