2.4 For PMN
Bacterial
Opsonophagocytic
Killing (OPH) Assay
1. Histopaque 1119 (Sigma 11191) and Histopaque 1077
(Sigma 10771).
2. 15- and 50-ml sterile plastic conical Falcon tubes.
3. Sterile petri dishes, 60 Â 15 mm.
4. Phosphate buffer saline (PBS, 1Â).
5. 2% Gelatin solution: Prepare a solution of 2% by dissolving 1 g
in 50 ml Hanks’ balanced salt solution without magnesium and
calcium (HBSSÀ). Bring to a boil and place at 37
C in 50 ml
tube until use. Should be used within a week of preparation.
6. +++ and ÀÀ+ buffers: HBSS+ and HBSSÀ respectively supplemented with 0.1% gelatin (dilute 1 ml of 2% gelatin in 19 ml
corresponding HBSS). Prepare fresh each time.
7. RBC Lysis buffer: Dissolve 8.29 g of NH 4 Cl (ammonium
chloride), 1 g of NaHCO 3 (sodium bicarbonate) and 0.038 g
of EDTA in a final volume of 1 L of distilled water. Filtersterilize the buffer and store it at 4
C.
8. RP-10 medium: RPMI-1640 medium supplemented with 10%
heat-inactivated FBS.
9. RP-10 supplemented with 2 mM EDTA: Add 20 ml of 50 mM
EDTA prepared in 1Â PBS to a volume of 480 ml of RP-10
medium.
10. 10 ml syringe and 25-G needle.
11. Non-tissue culture treated flat bottom 96-well plate.
2.5 General
Equipment
1. Incubator at 37
C + 5% CO 2 .
2. 37
C shaker.
3. Tabletop centrifuge (we use the Eppendorf Centrifuge 5418
model to spin the bacteria at 1700 rcf and Microtainer tubes at
7607 rcf).
4. Benchtop centrifuge (we use the Beckman Coulter Allegra™
6R model to centrifuge PMNs at 328 rcf at 4
C).
Fig. 1 Schematic depicting assay procedures
562
Manmeet Bhalla et al.
Bacterial
Opsonophagocytic
Killing (OPH) Assay
1. Histopaque 1119 (Sigma 11191) and Histopaque 1077
(Sigma 10771).
2. 15- and 50-ml sterile plastic conical Falcon tubes.
3. Sterile petri dishes, 60 Â 15 mm.
4. Phosphate buffer saline (PBS, 1Â).
5. 2% Gelatin solution: Prepare a solution of 2% by dissolving 1 g
in 50 ml Hanks’ balanced salt solution without magnesium and
calcium (HBSSÀ). Bring to a boil and place at 37
C in 50 ml
tube until use. Should be used within a week of preparation.
6. +++ and ÀÀ+ buffers: HBSS+ and HBSSÀ respectively supplemented with 0.1% gelatin (dilute 1 ml of 2% gelatin in 19 ml
corresponding HBSS). Prepare fresh each time.
7. RBC Lysis buffer: Dissolve 8.29 g of NH 4 Cl (ammonium
chloride), 1 g of NaHCO 3 (sodium bicarbonate) and 0.038 g
of EDTA in a final volume of 1 L of distilled water. Filtersterilize the buffer and store it at 4
C.
8. RP-10 medium: RPMI-1640 medium supplemented with 10%
heat-inactivated FBS.
9. RP-10 supplemented with 2 mM EDTA: Add 20 ml of 50 mM
EDTA prepared in 1Â PBS to a volume of 480 ml of RP-10
medium.
10. 10 ml syringe and 25-G needle.
11. Non-tissue culture treated flat bottom 96-well plate.
2.5 General
Equipment
1. Incubator at 37
C + 5% CO 2 .
2. 37
C shaker.
3. Tabletop centrifuge (we use the Eppendorf Centrifuge 5418
model to spin the bacteria at 1700 rcf and Microtainer tubes at
7607 rcf).
4. Benchtop centrifuge (we use the Beckman Coulter Allegra™
6R model to centrifuge PMNs at 328 rcf at 4
C).
Fig. 1 Schematic depicting assay procedures
562
Manmeet Bhalla et al.