An Assay to Assess Phagocytosis and Oxidative Burst Activity In Granulocytes and Monocytes

Overview

This video demonstrates the assay to confirm phagocytic activity by cells in human blood via flow cytometry. The granulocytes and monocytes confirm their phagocytic activity by showing green and red fluorescence confirming the presence of engulfed bacteria and oxidative burst activity respectively.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Assay Preparation

1. Gather, prepare, and organize the materials specified for the procedure (please refer to Table of Specific Materials/Equipment).
   1. Label 12 x 75 mm tubes.
      1. Label two tubes for each participant: one tube for the phagocytosis assay (label this tube with black ink and an "F" for fluorescein isothiocyanate [FITC]) and one tube for the oxidative burst (OB) activity assay (label this tube with red ink and an "H" for dihydroethidium [HE]).      
         NOTE: The use of sterile 12 x 75 mm tubes is recommended to minimize unintended cellular activation and associated increase in background noise.
      2. Label three tubes for the assay controls: one tube for the phagocytosis assay (FITC control: label this tube with black ink and an "F"), one tube for the OB activity assay (HE control: label this tube with red ink and an "H"), and one tube for the negative control (Blood only: label this tube with green ink and "blood only").
   2. Prepare stock solutions at least one day before the day of the assay.
      1. Prepare the sterile phosphate-buffered saline (PBS). Dilute 100 ml of 10x PBS with 900 ml of 18.2 MΩ H₂O. Filter sterilize with a 0.2 µm filter. Store at 4 °C until use.
      2. Prepare the sterile PBS glucose. Dissolve 1.0 g of glucose in 100 ml of PBS. Filter sterilize with a 0.2 µm filter. Store at 4 °C until use.
      3. Prepare the sterile 0.1 M citrate buffer. Dissolve 1.2 g of citric acid and 1.1 g of sodium citrate in 100 ml of 18.2 MΩ H₂O. Adjust the pH to 4.0. Filter sterilize with a 0.2 µm filter. Store at 4 °C until use.
      4. Prepare the HE stock solution. Resuspend 1.0 mg of HE in 1.0 ml of dimethyl sulfoxide (DMSO). Mix gently, aliquot, and store at -20 °C until use.
      5. Prepare the FITC-labeled Staphylococcus aureus (S. aureus) stock solution (2 x 10⁹ particles/ml). Resuspend 10 mg of FITC-labeled S. aureus in 1.5 ml (or appropriate amount) of PBS. Divide into three 500 µl aliquots and sonicate according to the manufacturer's recommendation. Aliquot and store at -20 °C until use.
      6. Prepare the unlabeled S. aureus stock solution (2 x 10⁹ particles/ml). Resuspend 100 mg of unlabeled S. aureus in 15 ml (or appropriate amount) of PBS. Divide into thirty 500 µl aliquots and sonicate according to the manufacturer's recommendation. Aliquot and store at -20 °C until use.
   3. Prepare fresh working solutions on the day of the assay.
      1. Prepare the HE working solution. Transfer 10 µl of thawed HE stock solution to 990 µl of PBS-glucose. Protect from light and keep on ice until use.
      2. Prepare the FITC-labeled S. aureus working solution (133,333 particles/µl). Transfer 70 µl of thawed FITC-labeled S. aureus stock solution to 980 µl of PBS. Protect from light and keep on ice until use.
      3. Prepare the unlabeled S. aureus working solution (133,333 particles/µl). Transfer 70 µl of thawed unlabeled S. aureus stock solution to 980 µl of PBS. Protect from light and keep on ice until use.
      4. Prepare the quench solution. Add 750 µl of 0.4% trypan blue solution to 11.25 ml of sterile 0.1 M citrate buffer. Keep in an ice-water bath until use.
   4. Have a certified phlebotomist draw blood according to the World Health Organization's guidelines for drawing blood.
      1. Draw blood into one 4 ml blood collection tube containing dipotassium ethylenediaminetetraacetic acid (K₂EDTA). Invert the blood collection tube according to the manufacturer's instructions. Keep the blood collection tube at room temperature on a bench-top rocker until use. Use this blood for the complete blood count (CBC) with white blood cell (WBC) differential analysis described in Step 1.2.1.
      2. Draw blood into one 4 ml blood collection tube containing lithium heparin. Invert the blood collection tube according to the manufacturer's instructions. Keep the blood collection tube at room temperature (RT) on a bench-top rocker until use. Use this blood for the monocyte and granulocyte phagocytosis and OB activity assay described in Step 2.
2. Determine the amount of bacteria (i.e., S. aureus) that will be needed to complete the assay for each sample.
   1. Use a hematology analyzer to perform a CBC with WBC differential analysis on the blood as described in the manufacturer's instructions. Make a note of the WBC count (in cells/ml), %Neutrophil, and %Monocyte values.
   2. Use the equations below to determine the neutrophil and monocyte counts for each sample.        
      Neutrophil count (in cells/ml) = %Neutrophil × WBC (in cells/ml)
      Monocyte count (in cells/ml) = %Monocyte × WBC (in cells/ml)
   3. Use the equations below to determine the phagocyte count for each sample and the number of phagocytes present in 100 µl of the sample.
      Phagocyte count (in cells/ml) = Neutrophil count (in cells/ml) + Monocyte count (in cells/ml)  
      Number of phagocytes in 100 µl = (Phagocyte count (in cells/ml))/10
   4. Use the equation below to determine the number of bacteria (i.e., S. aureus) needed for each sample and the amount of FITC-labeled S. aureus or unlabeled S. aureus working solution (133,333 particles/ml) that should be added to each tube.     
      NOTE: The target bacteria: phagocyte ratio is 8:1.
      Number of bacteria needed = Number of phagocytes in 100 µl × 8
      Volume of FITC or unlabeled working solution needed (in µl) = (number of bacteria needed)/(133,333 particles/µl)

2. Perform Assay

1. Prepare whole blood for the assay.
   1. For each participant and control tube, use an extended-length pipette tip to transfer 100 µl of whole blood from the lithium heparin blood collection tube to the bottom of an appropriately labeled 12 x 75 mm tube. Replace caps.
      NOTE: Be careful to avoid getting blood on the sides of the 12 x 75 mm tubes.
   2. Use a micropipette to add 10 µl of HE working solution to the tubes labeled with red ink and an "H", including the HE control tube. Replace caps and vortex briefly.
   3. Incubate all tubes in a 37 °C water bath for 15 min. Then, immediately transfer all tubes to an ice-water bath for 12 min.   
      NOTE: Use an open metal rack and gently shake the rack every 5 min to ensure that the tubes are quickly and adequately warmed or chilled.
2. Add bacteria (i.e., S. aureus) to the sample and control tubes.
   1. Use a micropipette to add the appropriate amount (calculated in Step 1.2.4) of unlabeled S. aureus working solution to the tubes labeled with red ink and an "H", including the HE control tube. Replace caps and vortex briefly.
   2. Use a micropipette to add the appropriate amount (calculated in Step 1.2.4) of FITC-labeled S. aureus working solution to the tubes labeled with black ink and an "F", including the FITC control tube. Replace caps and vortex briefly.
   3. Vortex the "blood only" control tube, but do not add either type of bacteria (i.e., S. aureus) to it.
   4. Incubate all tubes in a 37 °C water bath for 20 min. Use an open metal rack and gently shake the rack every 5 min to ensure that the tubes are quickly and adequately warmed. After the incubation period, immediately transfer all tubes to an ice-water bath for 1 min.
3. Wash the cells.
   1. Use a repeater pipette to add 100 µl of ice-cold quench solution to all tubes. Replace caps, vortex briefly, and then incubate all tubes on ice for 1 min.
   2. Use a repeater pipette to add 1 ml of ice-cold PBS to all tubes. Vortex briefly, and then add an additional 2 ml of ice-cold PBS to all tubes and replace caps. Centrifuge all tubes for 5 min at 4 °C and 270 x g, and then aspirate the supernatant.
   3. Repeat Step 2.3.2 one time (total of 2 washes).
4. Prepare samples for flow cytometry.
   1. Use a repeater pipette to add 50 µl of ice-cold fetal bovine serum to all tubes. Briefly vortex all tubes.
   2. Transfer all tubes to an appropriate carousel and use an automated cell lyse preparation workstation with red blood cell (RBC) lysing and WBC fixing reagents to lyse the RBCs and fix the WBCs as described in the manufacturer's instructions.

Materials

Name	Company	Catalog Number	Comments
12 x 75 mm tubes, with cap	VWR	20170-579	You will need 2 tubes per sample ("H" and "F") plus 3 tubes per batch (for assay controls; "F", "H", and "Blood only").
PBS (10X), pH 7.4	Life Technologies	70011-044
Bottle top 0.2 µm cellulose acetate filter (500 ml capacity)	Fisher Scientific	09-741-07
Glucose, powder	Life Technologies	15023-021
Citric acid (anhydrous, cell culture tested, plant cell culture tested)	Sigma-Aldrich	C2404-100G
Sodium citrate tribasic dihydrate	Sigma-Aldrich	S4641-25G
Dihydroethidium (Hydroethidine) (HE)	Life Technologies	D11347
Dimethyl sulfoxide (DMSO) Anhydrous	Life Technologies	D12345
Staphylococcus aureus (Wood strain without protein A) BioParticles, fluorescein conjugate (FITC)	Life Technologies	S2851
Staphylococcus aureus (Wood strain without protein A) BioParticles, unlabeled	Life Technologies	S-2859
Trypan Blue Solution, 0.4%	Life Technologies	15250-061
4.0 mL vacutainer containing 7.2 mg K2EDTA, spray-dried	VWR	BD367861	You will need 1 K2 EDTA blood collection tube per sample.
4.0 mL vacutainer containing 68 USP units Lithium Heparin, spray-coated	VWR	BD367884	You will need 1 Lithium Heparin blood collection tube per sample.
COULTER Ac·T 5diff CP	Beckman Coulter	6605705	i.e., hematology analyzer
COULTER Ac·T 5diff Rinse	Beckman Coulter	8547167
COULTER Ac·T 5diff Fix	Beckman Coulter	8547171
COULTER Ac·T 5diff WBC Lyse	Beckman Coulter	8547170
COULTER Ac·T 5diff Hgb Lyse	Beckman Coulter	8547168
COULTER Ac·T 5diff Cal Calibrator	Beckman Coulter	7547175
COULTER Ac·T 5diff Control Plus	Beckman Coulter	7547198
AcT 5diff Diluent	Beckman Coulter	8547169
200 µL extended-length pipette tips	VWR	37001-526
Insulated ice pan	VWR	89198-980	For ice water bath.
Open metal tube rack	VWR	60916-702
Fetal Bovine Serum	Life Technologies	26140-111
TQ-Prep Workstation	Beckman Coulter	6605429	i.e., automated cell lyse preparation workstation
ImmunoPrep Reagent System	Beckman Coulter	7546999	i.e., RBC lyse/WBC fix reagent system
COULTER CLENZ	Beckman Coulter	8546929