2.5.2 Cells and Viral
Maintenance
1. MDCK, 293T, DF1, and A549 cells.
2. Minimum essential medium (MEM) (FOR MDCK, 293T, and
A549 cells).
3. Dulbecco’s modified Eagle medium (DMEM) (FOR DF1
cells).
4. 1% penicillin and streptomycin (P/S).
5. 10% fetal bovine serum (FBS).
6. Humidified incubator with 5% CO 2 at 37
C.
7. Virus strain: A/HK/1/68 clone, MA20C (HK68-MA20C;
H3N2), from E. Brown (University of Ottawa, Ottawa,
Ontario, Canada), WT BR59 virus.
8. Hemagglutinin (HA) and neuraminidase (NA) from A/Puerto
Rico/8/34 (H1N1) (PR8) or A/HK/1/68 (H3N2) (HK68).
9. Embryonated eggs.
2.5.3 Evaluation of Viral
Growth Kinetics
1. 1% P/S.
2. Tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin
(1 μg/ml for MDCK cells, 0.5 μg/ml for A549 cells).
2.5.4 In Vivo Mouse
Experiments
1. Specific-pathogen-free 4- to 9-week-old female BALB/c mice.
2.5.5 Immune Response
Assays
1. MiniCollect tubes (Greiner Bio-One).
2. Monoclonal antibodies (all from BioLegend):
(a) F4/80-phycoerythrin (PE).
(b) I-AE–peridinin chlorophyll protein (PerCP)–Cy5.5.
(c) CD11b-allophycocyanin (APC)-Cy7.
(d) Gr1-PE-Cy7.
(e) IA8-APC.
(f) CD11c-fluorescein isothiocyanate (FITC).
(g) DAPI (4
0 ,6-diamidino-2-phenylindole).
(h) CD3-APC.
(i) CD4-APC-Cy7.
(j) CD8-PerCP-Cy5.5.
(k) Dx5-FITC.
(l) γδT-PE.
(m) B220-PE-Cy7.
3. A549 cells.
4. RNeasy minikit (Qiagen).
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Maintenance
1. MDCK, 293T, DF1, and A549 cells.
2. Minimum essential medium (MEM) (FOR MDCK, 293T, and
A549 cells).
3. Dulbecco’s modified Eagle medium (DMEM) (FOR DF1
cells).
4. 1% penicillin and streptomycin (P/S).
5. 10% fetal bovine serum (FBS).
6. Humidified incubator with 5% CO 2 at 37
C.
7. Virus strain: A/HK/1/68 clone, MA20C (HK68-MA20C;
H3N2), from E. Brown (University of Ottawa, Ottawa,
Ontario, Canada), WT BR59 virus.
8. Hemagglutinin (HA) and neuraminidase (NA) from A/Puerto
Rico/8/34 (H1N1) (PR8) or A/HK/1/68 (H3N2) (HK68).
9. Embryonated eggs.
2.5.3 Evaluation of Viral
Growth Kinetics
1. 1% P/S.
2. Tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin
(1 μg/ml for MDCK cells, 0.5 μg/ml for A549 cells).
2.5.4 In Vivo Mouse
Experiments
1. Specific-pathogen-free 4- to 9-week-old female BALB/c mice.
2.5.5 Immune Response
Assays
1. MiniCollect tubes (Greiner Bio-One).
2. Monoclonal antibodies (all from BioLegend):
(a) F4/80-phycoerythrin (PE).
(b) I-AE–peridinin chlorophyll protein (PerCP)–Cy5.5.
(c) CD11b-allophycocyanin (APC)-Cy7.
(d) Gr1-PE-Cy7.
(e) IA8-APC.
(f) CD11c-fluorescein isothiocyanate (FITC).
(g) DAPI (4
0 ,6-diamidino-2-phenylindole).
(h) CD3-APC.
(i) CD4-APC-Cy7.
(j) CD8-PerCP-Cy5.5.
(k) Dx5-FITC.
(l) γδT-PE.
(m) B220-PE-Cy7.
3. A549 cells.
4. RNeasy minikit (Qiagen).
336
Dipasree Hajra et al.