2. Block the plate with 3% bovine serum albumin in TBS–Tween
20 (TBS-T) for 1 h at 22
C.
3. Dilute the isolated sera into TBS-T in ratios of 1:1000, 1:5000,
1:7500, and 1:10,000 and add to the plate.
4. Incubate the plates at 37
C with mild agitation for 3 h.
5. Add the secondary antibody (anti-mouse IgG, IgA, IgM
(H + L), IgE, highly X-adsorbed (biotin)) to the wells in a
1:1000 ratio and agitate for 2 h.
6. Add streptavidin to each well in a 1:1000 ratio and shake for
30 min.
7. Develop the signal with p-nitrophenylphosphate and the reaction using 0.75 M NaOH.
8. Quantify the signal using a plate reader spectrophotometer at
an absorbance of 405 nm.
9. Quantify the production of cytokines using IFN-γ and IL-17A
ELISA kits (R&D Systems).
3.9 Preparation
of Biofilm-Dispersed
Pneumococci
1. Propagate NCI-H292 epithelial cell line in T75 cell culture
flasks in RPMI-1640 medium at 37
C and 5% CO 2 . Approximately 5–10 flasks will be needed to generate a sufficient quantity of biofilms.
2. Incubate cultures at 37
C renewing the cell culture media
every 2 days.
3. Once the cells have reached 100% confluency, remove and
discard culture medium.
4. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM
EDTA solution to remove all traces of serum that contains
trypsin inhibitor.
5. Add 2.0–3.0 mL of trypsin–EDTA solution to flask and
observe cells under an inverted microscope until cell layer is
dispersed (usually within 5–15 min).
6. Add 6.0–8.0 mL of complete growth medium and aspirate cells
by gently pipetting.
7. Add 0.5 mL of cell suspension to each well of a 24-well cell
culture plate and incubate at 37
C and 5% CO 2 . As with the
T75 cell culture flasks, continue renewing cell culture media
every 2 days.
8. Once the cell layer has reached 100% confluency, remove and
discard the culture medium from the culture vessel.
9. Wash cell substratum three times with 1 mL PBS per well to
remove medium residues.
Hybrid Biological-Biomaterial Vector
471
20 (TBS-T) for 1 h at 22
C.
3. Dilute the isolated sera into TBS-T in ratios of 1:1000, 1:5000,
1:7500, and 1:10,000 and add to the plate.
4. Incubate the plates at 37
C with mild agitation for 3 h.
5. Add the secondary antibody (anti-mouse IgG, IgA, IgM
(H + L), IgE, highly X-adsorbed (biotin)) to the wells in a
1:1000 ratio and agitate for 2 h.
6. Add streptavidin to each well in a 1:1000 ratio and shake for
30 min.
7. Develop the signal with p-nitrophenylphosphate and the reaction using 0.75 M NaOH.
8. Quantify the signal using a plate reader spectrophotometer at
an absorbance of 405 nm.
9. Quantify the production of cytokines using IFN-γ and IL-17A
ELISA kits (R&D Systems).
3.9 Preparation
of Biofilm-Dispersed
Pneumococci
1. Propagate NCI-H292 epithelial cell line in T75 cell culture
flasks in RPMI-1640 medium at 37
C and 5% CO 2 . Approximately 5–10 flasks will be needed to generate a sufficient quantity of biofilms.
2. Incubate cultures at 37
C renewing the cell culture media
every 2 days.
3. Once the cells have reached 100% confluency, remove and
discard culture medium.
4. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM
EDTA solution to remove all traces of serum that contains
trypsin inhibitor.
5. Add 2.0–3.0 mL of trypsin–EDTA solution to flask and
observe cells under an inverted microscope until cell layer is
dispersed (usually within 5–15 min).
6. Add 6.0–8.0 mL of complete growth medium and aspirate cells
by gently pipetting.
7. Add 0.5 mL of cell suspension to each well of a 24-well cell
culture plate and incubate at 37
C and 5% CO 2 . As with the
T75 cell culture flasks, continue renewing cell culture media
every 2 days.
8. Once the cell layer has reached 100% confluency, remove and
discard the culture medium from the culture vessel.
9. Wash cell substratum three times with 1 mL PBS per well to
remove medium residues.
Hybrid Biological-Biomaterial Vector
471