CytoSelect™ 96-Well Phagocytosis Assay (Zymosan Substrate)

1. Zymosan Suspension : One 0.25 mL tube of prelabeled Zymosan in PBS, 5 X 108 particles/mL
2. Fixation Solution : One 2 mL tube of 3.2% Buffered Formaldehyde Solution
3. Blocking Reagent (100X) : One 50 μL tube
4. 10X Permeabilization Solution : One 0.5 mL tube of PBS/1% Triton X-100
5. Detection Reagent (250X) : One 20 μL tube
6. Detection Buffer : One 2 mL tube
7. Substrate : Two 1.5 mL tubes
8. Stop Solution : Two 1.5 mL tubes
9. Phagocytosis Inhibitor : One 20 μL amber tube of 2 mM Cytochalasin D in DMSO

1. Phagocyte and Culture Medium
2. PBS, PBS/0.1 % BSA
3. 37 °C Incubator, 5 % CO2 Atmosphere
4. Light Microscope
5. 96-well Microtiter Plate Reader

• Fully quantify phagocytosis with no manual cell counting
• High-throughput 96-well format
• Convenient quantitation in a standard microplate reader

• Zymosan Suspension: Thaw Zymosan suspension at 4 °C. Either nonopsonized or opsonized Zymosan particles can be used in phagocytosis assay. To opsonize Zymosan particles, incubate particles with desired serum or IgG for 30 minutes at 37 °C, pellet particles by centrifugation and wash a few times with sterile 1X PBS. Prior to using, resuspend the particles in the same volume of sterile 1X PBS. Store at 4 °C.
• 1X Blocking Reagent: Prepare the appropriate volume for the number of samples being tested. IMMEDIATELY prior to using, dilute the provided 100X Blocking Reagent 1:100 in 1X PBS/0.1 % BSA. Do not store.
• 1X Permeabilization Solution: Prepare the appropriate volume for the number of samples being tested. Prior to using, dilute the provided 10X Permeabilization Solution 1:10 in 1X PBS. Store at 4 °C. 4
• 1X Detection Reagent: Prepare the appropriate volume for the number of samples being tested. IMMEDIATELY prior to using, dilute the provided 250X Detection Reagent 1:250 in 1X PBS/0.1 % BSA. Do not store.

Note: This kit is suitable for adherent phagocytes only. For suspension cells, please use one of the following assay kits: • CytoSelect™ 96-well Phagocytosis Assay Kit (E.coli Substrate) #CBA-222 • CytoSelect™ 96-well Phagocytosis Assay Kit (Red Blood Cell Substrate) #CBA-220 The following assay protocol is written for a 96-well format. Refer to the below table for the appropriate dispensing volumes of other plate formats. Culture Dish 96-well 48-well 24-well Phagocyte Seeding 100 200 400 Volume (μL/well) Zymosan Suspension 10 20 40 (μL/well) Fix Solution (μL/well) 100 200 400 Permeabilzation Solution 100 200 400 (μL/well) Detection Buffer 50 100 200 (μL/well) Table 1: Dispensing Volumes of Different Plate Formats

I. Phagocytosis of Zymosan

1. Harvest and resuspend phagocytic cells in culture medium at 1 - 5 x 105 cells/mL or the appropriate concentration that yields 50-80 % confluency after overnight incubation. Seed 100 μL in each well of a 96-well plate and incubate overnight at 37 °C, 5 % CO2.
2. Treat phagocytes with desired activators or inhibitors.
3. Add 10 μL of Zymosan suspension to each well. Mix well and immediately transfer the plate to a cell culture incubator for 15 minutes - 2 hours. Each sample including a negative control without Zymosan particles should be assayed in duplicate.
4. Remove the culture medium by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times.
5. Gently add 200 μL of cold, serum-free medium (e.g. DMEM, RPMI) to each well. Promptly remove the cold media by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times. Repeat twice more. Note: For loosely attached cells, complete culture media is preferred to maintain cell attachment 5

II. Remove and block external particles Note: Perform steps with care, gently adding solutions as to not disrupt cell attachment

1. Add 100 μL of Fixation Solution to each well, incubating 5 minutes at room temperature.
2. Promptly remove the Fixation Solution by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times.
3. Wash twice with 1X PBS.
4. Add 100 μL of prediluted 1X Blocking Reagent to each well (see Preparation of Reagents Section). Incubate the plate for 60 minutes at room temperature on an orbital shaker.
5. Promptly remove the Blocking Reagent by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times. Wash three times with 1X PBS.

III. Detection of internalized particles Note: Perform steps with care, gently adding solutions as to not disrupt cell attachment

1. Remove the PBS wash and add 100 μL of prediluted 1X Permeabilization Solution (see Preparation of Reagents Section) to each well, incubate 5 minutes at room temperature.
2. Promptly remove the Permeabilization Solution by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times. Wash once with 1X PBS.
3. Add 100 μL of prediluted 1X Detection Reagent to each well (see Preparation of Reagents Section). Incubate the plate for 60 minutes at room temperature on an orbital shaker.
4. Promptly remove the Detection Reagent Solution by gently aspirating or inverting the plate and blotting on a paper towel. Gently tap several times. Wash three times with 1X PBS.
5. Add 50 μL of Detection Buffer to each well. Incubate the plate for 10 minutes at room temperature on an orbital shaker.
6. Initiate the reaction by adding 100 μL of Substrate. Incubate for 5-20 minutes at 37 °C.
7. Stop the reaction by adding 50 μL of the Stop Solution and mix by placing the plate on an orbital plate shaker for 30 seconds.
8. Read the absorbance of each well at 405 nm.