assessment of vaccine efficacy (Fig. 1). This protocol provides
details of methods to produce immune sera in a mouse model,
quantify the ability of antibodies to bind bacterial surfaces by flow
cytometry and subsequently evaluate the opsonophagocytic capacity of immune sera using bacterial killing assays by primary bone
marrow-derived mouse PMNs.
2 Materials
2.1 For Immune/
Hyperimmune Sera
Generation
1. Appropriate mice strain. We routinely produce immune sera
using male and female 8- to 12-weeks-old C57BL/6 mice
(Jackson Laboratories, USA), housed in a fully certified Lab
Animal Facility (LAF). All experiments are performed in accordance with the Laboratory Animals Welfare Act and the Guide
for the Care and Use of Laboratory Animals.
2. Vaccine: Prevnar
® (Wyeth pharmaceuticals Inc., USA) or
Pneumovax
® (Merck and Co Inc., USA).
3. Dissection tools: Suitable forceps, fine tipped scissors, dissection board.
4. 1 ml syringe; 27- and 18-G needles.
5. Microtainer tubes for blood collection.
2.2 For Bacterial
Cultures
1. Bacteria: S. pneumoniae TIGR4 strain is used for all the methods outlined in this chapter. Other strains of interest can also
be used.
2. Liquid media: Todd-Hewitt broth (Bacto™ BD 249240) and
Oxyrase
® (Oxyrase OB0050).
3. Blood agar plates (Tryptic soy agar supplemented with 5%
sheep blood).
2.3 For Bacterial
Binding Assays
1. FACS buffer: Hanks’ balanced salt solution with magnesium
and calcium (HBSS+) with 1% (wt/vol) heat-inactivated fetal
bovine serum (FBS) and 0.1% (wt/vol) sodium azide. Add 5 ml
FBS and 500 mg of sodium azide to a total of 495 ml of HBSS
+. Filter-sterilize the buffer and store it at 4
C for up to 1 year.
2. APC-conjugated anti-mouse IgG antibody (Polyclonal antibody, Invitrogen 17-4010-82).
3. 5 ml polystyrene round bottom 12 Â 75 mm FACS tubes.
4. Non-tissue culture treated round bottom 96-well plate.
5. Flow cytometer. We have a BD Fortessa™ in our facilities. Any
other cytometer can also be used.
6. Flow cytometry analysis software. We use FlowJo
® for analysis.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
561
details of methods to produce immune sera in a mouse model,
quantify the ability of antibodies to bind bacterial surfaces by flow
cytometry and subsequently evaluate the opsonophagocytic capacity of immune sera using bacterial killing assays by primary bone
marrow-derived mouse PMNs.
2 Materials
2.1 For Immune/
Hyperimmune Sera
Generation
1. Appropriate mice strain. We routinely produce immune sera
using male and female 8- to 12-weeks-old C57BL/6 mice
(Jackson Laboratories, USA), housed in a fully certified Lab
Animal Facility (LAF). All experiments are performed in accordance with the Laboratory Animals Welfare Act and the Guide
for the Care and Use of Laboratory Animals.
2. Vaccine: Prevnar
® (Wyeth pharmaceuticals Inc., USA) or
Pneumovax
® (Merck and Co Inc., USA).
3. Dissection tools: Suitable forceps, fine tipped scissors, dissection board.
4. 1 ml syringe; 27- and 18-G needles.
5. Microtainer tubes for blood collection.
2.2 For Bacterial
Cultures
1. Bacteria: S. pneumoniae TIGR4 strain is used for all the methods outlined in this chapter. Other strains of interest can also
be used.
2. Liquid media: Todd-Hewitt broth (Bacto™ BD 249240) and
Oxyrase
® (Oxyrase OB0050).
3. Blood agar plates (Tryptic soy agar supplemented with 5%
sheep blood).
2.3 For Bacterial
Binding Assays
1. FACS buffer: Hanks’ balanced salt solution with magnesium
and calcium (HBSS+) with 1% (wt/vol) heat-inactivated fetal
bovine serum (FBS) and 0.1% (wt/vol) sodium azide. Add 5 ml
FBS and 500 mg of sodium azide to a total of 495 ml of HBSS
+. Filter-sterilize the buffer and store it at 4
C for up to 1 year.
2. APC-conjugated anti-mouse IgG antibody (Polyclonal antibody, Invitrogen 17-4010-82).
3. 5 ml polystyrene round bottom 12 Â 75 mm FACS tubes.
4. Non-tissue culture treated round bottom 96-well plate.
5. Flow cytometer. We have a BD Fortessa™ in our facilities. Any
other cytometer can also be used.
6. Flow cytometry analysis software. We use FlowJo
® for analysis.
Testing Anti-Pneumococcal Antibody Function Using Bacteria and Primary. . .
561