Kinetic Assay to Measure NADPH Oxidase Activity using Flow Cytometry.
Terri Thayer and Clayton E. Mathews
Bone Marrow Removal
1) Pour 6.5 ml Hanks’ Balanced Salt Solution with Phenol Red (HBSS, Cambrex 10-508) per
plate into 15 ml conical tube.
- Label as bones and marrow
- Keep everything sterile
2) In hood:
A) Sacrifice mice with CO2.
B) Excise hind limbs and clean off as much tissue as possible.
C) Flush out marrow with several washings into marrow plate with the HBSS in the Marrow
plate using a 10mL syringe and a 26 G needle.
D) Keep marrow on ice
3) Spin down cells 300 x g for 5 min at 5-10°C.
Removing red blood cells from marrow
1) Ficoll gradient is made up of: 31ml Histopaque 1119 (Sigma) and 13 ml HBSS
2) Slowly remove supernatant by aspiration and then resuspend cells in 500 uL of cold Ficoll
gradient. Slowly add cold Ficoll gradient to 5 ml.
3) Centrifuge 300 x g for 10 min at 5-10°C.
4) Take off top layer (~2.5-3 ml) with a pipet and transfer into a new 15 ml conical tube, being
careful not to disturb the layers. Discard bottom layers.
5) Wash Cells with 7 mL of “Modified Clear HBSS” (mc-HBSS). Total volume ~10 mL
Modified clear HBSS (mc-HBSS)
Hanks’ Balanced Salt Solution without Phenol Red (Cambrex 10-527)
0.1% BSA
1mM HEPES
6) Centrifuge 300 x g for 5 min at 5-10°C.
7) Aspirate off "HBSS" and resuspend the cells in 2 ml of “Modified Clear HBSS” (mc-HBSS)
8) Count cells and dilute to 5 million cells / mL with mc-HBSS.
If cells are too dilute centrifuge (300 x g for 5 min at 5-10°C) and Q.S. to the correct volume
9) Keep on ice.
Flow Cytometery Assay
1) In 5ml round bottom polystyrene tubes (Falcon 12x75mm #352085):
a) Controls. Add 100 µL of cells (5 x 105 cells) to each tube
i) Unstained
ii) GR1-PerCp Cy 5.5. Stain with GR1-PerCp/Cy 5.5
iii) CD11b-APC. Stain with CD11b-APC
iv) GR1-PerCp Cy 5.5, CD11b-APC & DHR123: Stain with GR1-PerCp/Cy5.5 and
CD11b-APC
v) GR1-PerCp Cy 5.5, CD11b-APC, DHR123 & PMA: Stain with GR1-PerCp/Cy5.5
and CD11b-APC
b) Samples: Add 300 µL of cells (1.5 x 106 cells) and stain with GR1-PerCp/Cy5.5
and CD11b-APC
Currently, dilutions for antibodies are anti-mouse GR1-PerCp Cy 5.5 1:192 (Final) and antimouse CD11b-APC 1:384 (Final). Both antibodies are from BDPharmingen.
2) Incubate tubes for 30 min at 4°C
3) Wash with 2 ml mc-HBSS
4) Centrifuge 300 x g for 5 min at 5-10°C
5) Pour off HBSS and resuspend control tubes are resuspended in 250 µL mc-HBSS and
resuspend all sample tubes with in 1 ml mc-HBSS
6) Labeling with Dihydrorhodamine 123 (DHR 123,Molecular Probes)
a) Stock concentration of DHR 123 is 100 µM.
b) DHR 123 is not added to the first 3 control tubes above (#1, 2, and 3)
c) Add 4µL of 100µM DHR 123 is added to Control tubes #4 and 5
d) To the Samples, add 10µL of 100µM DHR 123
Incubate at 37°C for 5 min.
7) Store cells on ice.
8) Add 4 µL of 10µM Phorbol 12-myristate 13-acetate (PMA, Sigma) to control tube #5.
Incubate control tubes # 4 and 5 for 15 min at 37°C
9) Calibrate Flow cytometer using the 5 control tubes
10) Remove 200 µL from each sample tube add to a 12x75mm FACS tube and assay basal
DHR123 fluorescence (time zero).
Just prior to placing cells on the FACS, vortex tube briefly to suspend cells. This is speed
the counting process.
11) Add PMA to cells at timed intervals. 10 µL of 10 µM PMA is added to the tubes at timed
intervals and the tubes are placed in a 37°C water bath for the remainder of the assay. If
PMA is added every minute, then 5 tubes can be run at a time. If PMA is added every 45
seconds, then 6 tubes can be run.
12) At 5, 10, 20, and 30 min., 200 uL of cells are removed and assayed.