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Macrophage Polarization and Phagocytosis Assay
Instructions for use

Safety Statements
These products are not for use in GMP
manufacturing, nor human or animal in vivo use,
including use as a diluent, as an excipient, or for
diagnostic use.
These products are for research use only.
WARNING: LONZA PRIMARY CELLS CONTAINHUMAN SOURCE MATERIAL; TREAT AS
POTENTIALLY INFECTIOUS. Each donor is tested
and found non-reactive by an FDA-approved method
for the presence of HIV-I, hepatitis B virus andhepatitis C virus. Where donor testing is notpossible, cell products are tested for the presence ofviral nucleic acid from HIV-1, hepatitis B virus, andhepatitis C virus. Testing cannot offer complete
assurance that HIV-1, hepatitis B virus, and hepatitis
C virus are absent. All human-sourced products
should be handled at the biological safety level 2 tominimize exposure to potentially infectious products,
as recommended in the CDC-NIH manual, Biosafetyin Microbiological and Biomedical Laboratories, 5thedition. If you require further information, pleasecontact your site safety officer or Scientific Support.
Preparation of Reagents
All work should be performed in a laminar flow hood.
Decontaminate the external surfaces of all
supplement vials and the medium bottles with ≥ 70%
ethanol or isopropanol.
1. KuGM™ Modified Medium
a. Add the 50 mL FBS supplement to the500 mL KuBM™ basal medium as permanufacturer instructions.
b. Only add 500 µL of the GA-1000
supplement. DO NOT ADD THE WHOLE
SUPPLEMENT.

2. M-CSF Macrophage Differentiation Medium
a. Add 1 mL of PBS into M-CSF 10 µg stock
vial and mix well (makes 10 µg/mL workingsolution).
b. Combine KuGM™ Modified Medium
prepared in step 1 + 350 µL M-CSF
10 µg/mL working solution to make 100 mLof 35 ng/mL Macrophage Differentiation
Medium.
3. M1 Polarization Medium
a. Prepare this medium on day 6 of culture(see PART 3: Macrophage Polarization
below).
b. IFN-γ 100 µg/mL solution: Add 0.8 µL
0.1% HSA to 200 µL PBS. Then add thissolution to the 20 µg lyophilized vial ofIFN-γ.
c. LPS 100 µg/mL solution: Add 10 µL of
1 mg/mL stock LPS solution to 90 µL
KuGM™ Modified Medium.
d. For each 10 mL of M1 Polarization
Medium: combine 5 µL IFN-γ solution+ 1 µL LPS solution + 10 mL of KuGM™
Modified Medium.
4. M2 Polarization Medium
a. Prepare this medium on day 6 of culture(see PART 3: Macrophage Polarization
below).
b. IL-4 100 µg/mL solution: Add 0.8 µL 0.1%
HSA to 200 µL PBS. Then add this solution
to the 20 µg lyophilized vial of IL-4.
c. IL-10 100 µg/mL solution: Add 0.4 µL 0.1%
HSA to 100 µL PBS. Then add this solution
to the 10 µg lyophilized vial of IL-10.
d. For each 10 mL of M2 Polarization
Medium: combine 2 µL IL-4 solution + 2 µL
IL-10 solution + 10 mL KuGM™ Modified
Medium.

Page | 1 of 4

5. Cytochalasin D Medium
a. Prepare on day 7 of culture (see PART 4:
Phagocytosis Assay using Live CellImaging).
b. Reconstitute lyophilized cytochalasin D(1 mg vial) to 4 mM stock solution by adding
493 µL DMSO to the vial and mixing well.
c. 4 µM Cytochalasin D Medium: prepare
1 mL of medium by adding 1 µL of 4 mM
cytochalasin D stock solution to 1 mL
KuGM™ Modified Medium. Scale to total
volume needed.
Note about this protocol: There are two possible
starting points for the macrophage differentiation
protocol.
1. Part 1 describes the protocol if using
cryopreserved PBMCs as the starting material,from which monocytes are first isolated via
plastic adherence before being treated withdifferentiation medium. Upon completing thisPart, you would skip Part 2 and move directly toPart 3.
2. Part 2 describes the protocol if using
cryopreserved positively-selected CD14+
Monocytes as the starting material. Skip Part 1
if using Monocytes as the starting material.
Part 1: Macrophage Differentiation Protocol
Using PBMCs as Starting Material (Option 1)
NOTE: Perform all work in a laminar flow hood.
NOTE: Cryopreserved cells are delicate and must
be thawed and put into culture as quickly as possible
with minimal handling. Thaw no more than 2 vials ata time.
NOTE: Follow these steps only if using Peripheral
Blood Mononuclear Cells (PBMCs) as a startingmaterial.
1. Add 10 mL of pre-warmed (37°C) KuGM™
Modified Medium to a 15 mL conical centrifugetube.
2. Thaw PBMC vials (Lonza part no. CC-2702,
50M cells) according to manufacturer
instructions in a 37°C water bath for no morethan 2 minutes, removing cryo vials once a sliverof ice remains.
a. Other vialing densities can be used as
appropriate to your workflow.
3. Transfer PBMCs to the 15 mL conical tube
containing KuGM™ Modified Medium.
4. Centrifuge tubes at 300xg for 10 minutes atroom temperature.
5. Remove supernatant and discard.

6. Resuspend cells in 5 mL KuGM™ ModifiedMedium.
7. Count using trypan blue and a hemocytometerat a 1:100 dilution
a. Example: First, dilute cells 1:10 KuGM™
Modified Medium (10 µL cells + 90 µL
medium, e.g.). Then, dilute again 1:10 in
0.4% trypan blue (e.g., 10 µL diluted cells +90 µL 0.4% trypan blue).
8. Add KuGM™ Modified Medium to the 15 mL
conical tube to resuspend PBMCs at a density of
10x106 cells/mL.
9. Seed cells at the following densities:
a. For a 24-well plate, add 1 mL of cellsuspension for a total of 10x106 cells/well.
b. For a 96-well plate, add 100 µL of cellsuspension for a total of 1x106 cells/well.
10. Incubate in a humidified incubator for 2 hours at37°C, 5% CO2.
11. Remove plate from incubator, swirl to gently
remove non-adhered cells, and aspirate media
from all wells.
12. Wash plate 2x with pre-warmed (37°C) KuGM™
Modified Medium.
13. Add M-CSF Macrophage Differentiation Medium
to each well:a. For a 24-well plate: 1 mL per wellb. For a 96-well plate: 100 µL per well
14. Replace plate in the incubator. Do not disturb
until day 3.
15. PROCEED TO PART 3.
Part 2: Macrophage Differentiation Protocol
Using Positively-selected CD14+ Monocytes as
Starting Material (Option 2)
NOTE: Perform all work in a laminar flow hood.
NOTE: Cryopreserved cells are delicate and must
be thawed and put into culture as quickly as possible
with minimal handling. Thaw no more than 2 vials ata time.
NOTE: Follow these steps only if using positivelyselected CD14+ Monocytes as a starting material.
1. Add 10 mL of pre-warmed (37°C) KuGM™
Modified Medium to a 15 mL conical centrifugetube.
2. Thaw CD14+ Monocytes (Lonza part no.
2W-400B; positively-selected) according to
manufacturer instructions in a 37°C water bathfor no more than 2 minutes, removing cryo vials
once a sliver of ice remains.
3. Transfer monocytes to the 15 mL conical tube
containing KuGM™ Modified Medium.
4. Centrifuge tubes at 300xg for 10 minutes atroom temperature.

Page | 2 of 45. Remove supernatant and discard.
6. Resuspend cells in 5 mL KuGM™ ModifiedMedium.
7. Count using trypan blue and a hemocytometerat a 1:20 dilution
a. Example: 10 µL cells + 190 µL 0.4% trypanblue
8. Add M-CSF Macrophage Differentiation Medium
to the 15 mL conical tube to resuspend CD14+
Monocytes at a density of 1x106 cells/mL.
9. Seed cells at the following densities:
a. For a 24-well plate, add 1 mL of cellsuspension for a total of 1x106 cells/well.
b. For a 96-well plate, add 100 µL of cellsuspension for a total of 0.1x106 cells/well.
10. Incubate in a humidified incubator at 37°C and
5% CO2. Do not disturb until day 3.
11. PROCEED TO PART 3.
Part 3: Macrophage Polarization
1. On day 3, change media by carefully aspirating
medium from all wells and replacing with theappropriate amount of M-CSF Macrophage
Differentiation Medium:a. For a 24-well plate: 1 mL per wellb. For a 96-well plate: 100 µL per well
2. On day 6, make M1 and/or M2 Polarization
Medium as described in the “Preparation ofReagents” section of this protocol.
3. Carefully aspirate medium from each well anddiscard.
4. For polarization, add the appropriate polarization
medium (M1, M2, or naïve control [M-CSFMacrophage Differentiation Medium]) to eachwell:a. For a 24-well plate: 1 mL per wellb. For a 96-well plate: 100 µL per well
5. Return to the incubator and culture for 24 hours.
NOTE: After 24 hours, polarization should haveoccurred (to M1 or M2 state, depending on whichpolarization medium was used). A variety ofanalyses or assays may be carried out at this point.
Proceed to PART 4 for an example of aphagocytosis assay using a live cell imaging system.
Part 4: Phagocytosis Assay Using Live CellImaging
NOTE: We used a Sartorius Incucyte® SX5 Live CellAnalysis Instrument to carry out this assay. Othersystems may be used, but may require some
optimization of instrument parameters and staining
protocol.

NOTE: We performed this assay in a clear, 96-wellflat-bottomed plate format using cytochalasin D as aphagocytosis inhibitor. Other well formats andcompounds can be tested, adjusting for volume andconcentration, following the same general workflow
as below.
1. Reconstitute pHrodo™ bioparticles: On day 7(24 hours after addition of M1/M2 Polarization
Medium), 2 hours prior to establishing theassay, reconstitute pHrodo™ green E. coli
bioparticles (2 mg vial) by adding 2 mL PBS,transferring mixture to a glass vial, andsonicating in a water bath for 15 minutes. Returnto the refrigerator until adding to cells.
NOTE: Reconstituted pHrodo™ bioparticles can be
stored at 4°C for up to one month.
2. Prepare cytochalasin D medium as describedin the “Preparation of Reagents” section above.
3. 1 hour prior to assay start, remove
polarization media from all wells.
4. Wash all wells 1x with KuGM™ ModifiedMedium.
5. Add 100 µL of KuGM™ Modified Medium to allwells that will serve as assay blanks (nopHrodo™ bioparticles, no treatment).
6. Add 75 µL of KuGM™ Modified Medium to alltreatment wells with pHrodo™ bioparticles.
7. Add 50 µL of KuGM™ Modified Medium and25 µL of 4 µM cytochalasin D medium (finalconcentration 1 µM per well) to negative controlwells. Incubate for 1 hour at 37°C and 5% CO2.
8. 30 minutes prior to assay, sonicate thepHrodo™ bioparticles in a water bath again for
15 minutes.
9. Dilute the pHrodo™ bioparticle suspension to
0.4 µg/µL by adding 1 mL of the pHrodo™
bioparticle suspension to 1.5 mL of KuGM™
Modified Medium in a separate glass vial.
10. Sonicate the diluted pHrodo™ bioparticle
suspension for 10 minutes in a water bath.
11. Vortex the diluted pHrodo™ bioparticle
suspension after dilution. Do this immediately
prior to adding to wells.
12. Add 25 µL of diluted pHrodo™ bioparticle
solution to all control and treatment wells(defined in steps #6 and #7 above). All wells
now contain 100 µL volume.
13. Immediately place into the incubator with live
cell imager and initiate image capture protocol.
a. We recommend 20x magnificationb. Color channel should be set to theappropriate fluorescent signal for thefluorescent tag used on the bioparticle(e.g., GFP green). Consult bioparticle
manufacturer instructions for moreinformation.

Page | 3 of 4

Ordering Informationc. 3x images per well recommendedd. The Incucyte® protocol used, captures 3
images per well every 15 minutes for 6hours, then every 1 hour for the next 18hours.
e. Total assay time = 24 hours.

Catalog No.

Description

Size

CC-2702

Cryopreserved
Human Peripheral
Blood
Mononuclear
Cells (HPBMC)≥50 million cells

Cryopreserved
Human Peripheral
Blood CD14+
Monocytes,
PositivelySelected≥40 million cells

KuGM™ Kupffer
Cell Culture
BulletKit®

500 mL Basal
medium +supplement kit

CC-2703
CC-2705
CC-2704

If performing the assay described above,
macrophages (especially M2 macrophages) treatedwith green GFP-tagged pHrodo™ bioparticles should
begin to fluoresce as they consume the bioparticles
over time, resulting in a stronger fluorescent signal
and should appear visually greener in the composite
videos. Macrophages treated with cytochalasin Dand pHrodo™ bioparticles should show inhibited
rates of consumption and far lower fluorescence.
Macrophages not treated with fluorescentbioparticles should show no fluorescence at all.

2W-400A
2W-400B
2W-400C

MKC-500BK≥100 million cells≥25 million cells≥10 million cells≥20 million cells≥10 million cells

Example videos can be found on the Lonza website.

Corning® 96-Well plate (Corning® 3595) mentioned is a product ofCorning®.
Corning® Falcon® 24-Well plate (Corning® 353047) mentioned is aproduct of Corning®.
Gibco® Trypan Blue 0.4% (ThermoFisher Scientific 15250061)
mentioned is a product of GIBCO®.
PBS without Calcium or Magnesium (ThermoFisher Scientific10010023) mentioned is a product of GIBCO®.
InvitrogenTM pHrodoTM Green E. coli BioParticlesTM Conjugate forPhagocytosis (FisherScientific P35366) mentioned is a product ofInvitrogenTM.
Cytochalasin D (Sigma C8273-1MG) mentioned is a product ofMilliporeSigma.
Recombinant Human M-CSF (PeproTech 300-25) mentioned is aproduct of PeproTech.
IFN-γ (PeptroTech 300-02) mentioned is a product of PeproTech.
LPS (Sigma L-5668) mentioned is a product of MilliporeSigma.
IL-4 (PeproTech 200-04) mentioned is a product of PeproTech.
IL-10 (PeproTech 200-10) mentioned is a product of PeproTech.

For research use only. Not for use in diagnostic procedures. All
trademarks belong to Lonza, and are registered in the USA, EU
and/or CH or belong to third-party owners and are used only for
informational purposes. All third-party copyrights have been
reproduced with permission from their owners. The information
contained herein is believed to be correct and corresponds to thelatest state of scientific and technical knowledge. However, nowarranty is made, either expressed or implied, regarding its
accuracy or the results to be obtained from the use of suchinformation and no warranty is expressed or implied concerning theuse of these products. The buyer assumes all risks of use and/orhandling. Any user must make his own determination and satisfy
himself that the products supplied by Lonza Group Ltd or its
affiliates and the information and recommendations given by Lonza
Group Ltd or its affiliates are (i) suitable for intended process or
purpose, (ii) in compliance with all applicable laws, including allenvironmental, health and safety regulations, and (iii) will not
infringe any third party’s intellectual property rights. The user bears
the sole responsibility for determining the existence of any suchthird-party rights, as well as obtaining any necessary licenses andapprovals. For more details: http://www.lonza.com/legal©2024 Lonza. All rights reserved.

Incucyte® SX5 Live Cell Analysis Instrument mentioned is a
product from Sartorius.]
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CD-OP027 05/24