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Protocol
Protocol to isolate and analyze mouse bone
marrow derived dendritic cells (BMDC)
Different types of immune cells are involved in atherogenesis and may act atheroprotective or
atheroprogressive. Here, we describe an in vitro approach to analyze CD11c
+
cells and CD11c
+
-
derived ApoE in atherosclerosis. The major steps include harvesting mouse bone marrow, plating
cells in culture dishes, treating them with differentiation factors, and collecting cells after removal
of undesirable populations. This protocol can be adapted for CD11c
+
cells in different contexts,
thus, serving as models for different diseases and to analyze cell-specific molecules.
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional
guidelines for laboratory safety and ethics.
Manuela Sauter,
Reinhard J. Sauter,
Henry Nording,
Marcus Olbrich,
Frederic
Emschermann,
Harald F. Langer
manuela.sauter@uksh.de
(M.S.)
harald.langer@uksh.de
(H.F.L.)
Highlights
Detailed protocol to
isolate bone-marrow-
derived dendritic
cells (BMDC)
Exposing BMDC to an
atherosclerotic
environment in vitro
Suggestions to
analyze BMDC in the
context of
atherosclerosis
Sauter et al., STAR Protocols 3,
101664
September 16, 2022 ª2022
The Author(s).
https://doi.org/10.1016/
j.xpro.2022.101664
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Protocol
Protocol to isolate and analyze mouse bone marrow
derived dendritic cells (BMDC)
Manuela Sauter,
1,4,
*Reinhard J. Sauter,
1
Henry Nording,
1
Marcus Olbrich,
2
Frederic Emschermann,
2
and Harald F. Langer
1,3,5,
*
1
Department of Cardiology, University Hospital, Medical Clinic II, University Heart Center Luebeck, Ratzeburger Allee 160,
23538 Luebeck, Germany
2
University Hospital, Department of Cardiology, Eberhard Karls University Tuebingen, 72076 Tuebingen, Germany
3
Department of Cardiology, Angiology, Haemostaseology and Medical Intensive Care, University Medical Centre Mannheim,
Medical Faculty Mannheim, Heidelberg University, 68167 Mannheim, Germany
4
Technical contact
5
Lead contact
*Correspondence: manuela.sauter@uksh.de (M.S.), harald.langer@uksh.de (H.F.L.)
https://doi.org/10.1016/j.xpro.2022.101664
SUMMARY
Different types of immune cells are involved in atherogenesis and may act athe-
roprotective or atheroprogressive. Here, we describe an in vitro approach to
analyze CD11c
+
cells and CD11c
+
-derived ApoE in atherosclerosis. The major
steps include harvesting mouse bone marrow, plating cells in culture dishes,
treating them with differentiation factors, and collecting cells after removal
of undesirable populations. This protocol can be adapted for CD11c
+
cells in
different contexts, thus, serving as models for different diseases and to analyze
cell-specific molecules.
For complete details on the use and execution of this protocol, please refer to
Sauter et al. (2021).
BEFORE YOU BEGIN
The protocol below describes the specific steps for the isolation and differentiation of CD11c
+
cells
from murine bone marrow and their exposure to an atherosclerotic environment in vitro. Experience
in cell culture techniques is required.
Cells isolated from bone marrow can also be differentiated to other cell types, depending on the
culture conditions and additives.
Institutional permissions
Experiments involving mice must conform to institutional and governmental guidance. The proced-
ures in this protocol were approved by the Regierungspraesidium Tuebingen (approvals M03/10
and M16/15) and performed in accordance with the Guide for the Care and Use of Laboratory
Animals and all other relevant ethical guidelines.
Preparation of cell culture medium
Timing: 30 min
STAR Protocols 3, 101664, September 16, 2022 ª2022 The Author(s).
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1
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1. Add supplements required for cell culture of BMDC to 430 mL fresh RPMI-medium:
2. pre-warm medium in water bath at 37C degrees.
DCmediumcanbestoredat4
C degrees for a maximum of 4 weeks. Check color of medium before
usage. If the color changes from yellowish red to purple-pink (which indicates pH changes), discard
the medium and make fresh one.
GM-CSF 20 ng/mL needs to be added freshly directly before cells are seeded into the 6 well plate.
Preparation of a 6 well plate for bone marrow isolation
Timing: 10 min
3. Add 70% Ethanol to the first well of the plate (to disinfect the bones).
4. Add approximately 1 mL medium to the second and to the third well (for washing the bones) and
3 mL medium to the fourth well of the plate (for flushing the bone).
KEY RESOURCES TABLE
DC medium
Reagent Final concentration Amount
RPMI – 1640 complete N/A 430 mL
Fetal Bovine Serum 10% (v/v) 50 mL
HEPES buffer 1 mM 5 mL
Penicillin Streptomycin 10,000 U/mL 1% (v/v) 5 mL
b-Mercaptoethanol 50 mM 1.8 mL
REAGENT or RESOURCE SOURCE IDENTIFIER
Experimental models: Organisms/strains
C57BL/6J; 6–8 week old female and male mice
can be used
The Jackson Laboratory Stock no. 000664; RRID:
IMSR_JAX:000664
Antibodies
CD103 PerCP/Cy5.5 clone 2E7 (Dilution: 3 mLin
100 mL FACS buffer)
BioLegend Cat. no. 121416; RRID:
AB_2128621
CD11b PE clone M1/70 (Dilution: 3 mL in 100 mL
FACS buffer)
BioLegend Cat. no. 101208; RRID:
AB_312791
CD11c APC clone N418 (Dilution: 3 mL in 100 mL
FACS buffer)
BioLegend Cat. no. 117310; RRID:
AB_313779
CD172a (SIRPa) PE/Dazzle594 clone P84
(Dilution: 3 mL in 100 mL FACS buffer)
BioLegend Cat. no. 144016
RRID: AB_2565280
CD45 PB clone 30-F11 (Dilution: 3 mL in 100 mL
FACS buffer)
BioLegend Cat. no. 103125; RRID:
AB_493536
F4/80 PE/Cyanine7 clone QA17A29 (Dilution:
3mL in 100 mL FACS buffer)
BioLegend Cat. no. 157307
RRID: AB_2832550
I-A/I-E MHCII FITC clone M5/114.15.2 (Dilution:
3mL in 100 mL FACS buffer)
BioLegend Cat. no. 107605; RRID:
AB_313320
IRDye800CW Goat anti-Rabbit IgG (Dilution:
1: 15.000)
LI-COR Cat. no. 926-32211; RRID:
AB_621843
Ly-6C Alexa Fluor700 clone 1A8 (Dilution: 3 mL
in 100 mL FACS buffer)
BioLegend Cat. no. 127621; RRID:
AB_10640452
Rabbit monoclonal to Apolipoprotein E (Dilution:
1: 5.000)
Abcam Cat. no. ab183596; RRID:
AB_2832971
TCR-b chain Brilliant Violet510 clone H57-597
(Dilution: 3 mL in 100 mL FACS buffer)
BioLegend Cat. no. 109233; RRID:
AB_2562349
(Continued on next page)
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2STAR Protocols 3, 101664, September 16, 2022
Protocol
Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Chemicals, peptides, and recombinant proteins
43Laemmli sample buffer Bio-Rad Cat. no. 1610747
Acetic acid Merck (Sigma-Aldrich) Cat. no. A6283
acetylated LDL Thermo Scientific Cat. no. L35354
Acrylamide RotiphoreseGel 30 Carl Roth Cat. no. 3029.1
APS Carl Roth Cat. no. 9592.3
Bovine Serum albumin fraction V Merck (Sigma-Aldrich) Cat. no. 10735094001
Coomassie Brilliant Blue R-250 protein stain powder Bio-Rad Cat. no. 1610400
Dulbecco’s PBS (DPBS) Gibco Cat. no. 14190-144
EDTA, 0.5 M, pH 8.0 Invitrogen Cat. no. AM9260G
Ethidium bromide solution Sigma-Aldrich Cat. no. E1510-10ML
Fetal bovine serum Thermo Scientific Cat. no. 26140-079
Formaldehyde solution 4%, buffered, pH 6.9 Merck Cat. no. 1004960700
Glycine Merck (Sigma-Aldrich) Cat. no. G8898-500G
HEPES buffer Gibco Cat. no. 11360-070
NaN
3
Merck (Sigma-Aldrich) Cat. no. 2002
Invitrogen Agarose UltraPureThermo Scientific Cat. no. 16500500
OneComp compensation beads BD Cat. no. 552845
PBS without Ca and Mg Gibco Cat. no. 14190-169
Penicillin and streptomycin Sigma-Aldrich Cat. no. P4333
RBC Lysis Buffer (eBioscience) Thermo Scientific Cat. no. 00-4333-57
Recombinant mouse GMCSF protein PeproTech Cat. no. 415-ML-010/CF
RPMI 1640 medium Thermo Scientific Cat. no. 11875-093
SDS blotting grade Carl Roth Cat. no. 0183.1
Skimmed milk powder AppliChem Cat. no. A0830
Streptavidin AP conjugate Merck (Roche) Cat. no. 11089161001
TEMED Carl Roth Cat. no. 2367.4
TRIS Carl Roth Cat. no. 4855.2
Tween-20 Merck (Sigma-Aldrich) Cat. no. P9416
X-Gal Merck (Sigma-Aldrich) Cat. no. 3117073001
Zombie NIRFixable Viability Kit, APC-Fire 750 BioLegend Cat. no. 423105
b-Mercaptoethanol Sigma-Aldrich Cat. no. M6250
Critical commercial assays
Cholesterol Efflux Kit (cell based) Abcam Cat. no. ab196985
Mouse Apolipoprotein E ELISA Kit Abcam Cat. no. ab215086
PierceBCA Protein Assay Kit Thermo Scientific Cat. no. 23225
Software and Algorithms
Cellquest Pro BD Biosciences https://www.bd.com/de-de/
products/biosciences
GraphPad Prism 9.2.0 GraphPad https://www.graphpad.com/
scientific-software/prism/
ImageJ Wayne Rasband, NIH https://imagej.nih.gov/ij/
Kaluza Analysis 2.1 Beckman Coulter https://www.beckman.de/flow-
cytometry/software/kaluza
Other
Cell strainer (70-mm pore size) Corning Cat. no. 431751
Dissecting scissors Fisher Scientific Cat. no. 08-940
Forceps Fisher Scientific Cat. no. 22-327379
Greiner centrifuge tubes, 50 mL, 30 3115 mm,
conical (V) bottom, w/ graduations, I.D. field
Merck Cat. no. T2318-500EA
Needle (27 gauge) BD Biosciences Cat. no. 305109
Petri dish Fisher Scientific Cat. no. 07-202-011
PVDF membranes (Immun-Blot PVDF Membrane) Bio-Rad Cat. no. 1620177
Serological glass pipettes 5 mL, 10 mL, 25 mL, 50 mL ROTILABO, Carl Roth N/A
Sterile syringe (10 mL) BD Biosciences Cat. no. 309604
Tissue culture plates (6 well) Corning Cat. no. 3516
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STAR Protocols 3, 101664, September 16, 2022 3
Protocol
MATERIALS AND EQUIPMENT
TBST buffer (TBS + 0.05% Tween-20)
To make 1 l of TBST wash buffer, add 100 mL of 103TBS and 500 mLTween20 detergent to 900 mL
of water.
Reducing Laemmli buffer
Add 100 mLofb-mercaptoethanol per 900 mL (final concentration of 355 mM).
For best results, do not store sample buffer with b-mercaptoethanol.
10% Polyacrylamide gels for SDS-PAGE
The required 10% separation gel was prepared as described in the following table. In this protocol,
4.5% stacking gels were used.
After pouring the separating gel solution into the gel cassettes, overcoat with isopropanol to ensure
a straight, smooth surface. After polymerization of the separating gels, remove the isopropanol,
pipette the freshly prepared collection gel solution onto it and insert a comb. After complete poly-
merization of the collection gel, place the gel cassettes in the electrophoresis chamber that is filled
with electrophoresis buffer. After removing the comb, pipette the samples into the gel pockets,
which should be rinsed with buffer beforehand.
DC medium
Reagent Final concentration Amount
RPMI – 1640 complete N/A 430 mL
Fetal Bovine Serum 10% (v/v) 50 mL
HEPES buffer 1 mM 5 mL
Penicillin Streptomycin 10,000 U/mL 1% (v/v) 5 mL
b-Mercaptoethanol 50 mM 1.8 mL
103TBS buffer
Reagent Final concentration Amount
TRIS 200 mM 24 g
NaCl 1500 mM 88 g
H
2
O N/A 900 mL
adjust the pH to 7.6 and final volume to 1 L.
10% separation gel
Reagent Final concentration Amount
30% (w/v) Acrylamide 10% 3.3 mL
1,5 M Tris-HCl pH 8.8 375 mM 2.5 mL
H
2
O N/A 4.1 mL
10% SDS 0.1% 100 mL
TEMED 0.09% 9 mL
10% APS 0.03% 27 mL
4.5% stacking gel
Reagent Final concentration Amount
30% (w/v) Acrylamide 4.5% 1.5 mL
0,5 M Tris-HCl pH 6.6 375 mM 2.5 mL
H
2
O N/A 5.9 mL
10% SDS 0.1% 100 mL
TEMED 0.2% 20 mL
10% APS 0.06% 60 mL
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4STAR Protocols 3, 101664, September 16, 2022
Protocol
STEP-BY-STEP METHOD DETAILS
Bone marrow cell isolation
Timing: 3 h
The following steps provide the procedure of collection and differentiation of bone marrow-derived
cells into BMDCs.
Note: Ensure that all the reagents and samples are kept on ice during the entire procedure.
Perform all steps in a laminar flow hood with sterile equipment to maintain sterility.
For one cell culture 6 well microplate, you will need bone marrow from one mouse.
Note: We have found that mice aged 6–8 weeks provide ideal conditions for cultivation of BM
derived CD11c
+
cells. Young mice provide a good amount of good quality BM cells, while
older mice provide more BM cells but the quality is not that good.
CRITICAL: Sterile technique is required for the isolation of BM cells; all tools should be
rinsed with ethanol, and procedures should be performed in a laminar flow hood.
1. Euthanize the mice withinhaled anesthetics (here, isoflurane was used: Inhalation of 5.0 relative vol. %
in an anesthesia chamber at an oxygen flow of 1 L/min) or CO
2
, followed by cervical dislocation.
2. Put the mouse abdomen down-faced on a towel sprayed with 70% ethanol. Sterilize the hind legs
and back by spraying with 70% (vol/vol) ethanol. Pin the paws to a suitable surface (Styrofoam for
example).
3. Using scissors, cut the midline of the back and continue cutting to expose the femurs (see Figure 1A).
4. Unpin the right leg and peel the skin off toward the midline.
a. Cut the adductor muscles toward the midline and sever the leg between the hip joint and the
spine (see Figure 1B).
b. Once the leg is detached from the body, separate the femur from the tibia by cutting the knee
joint.
c. Cutawaythepawandremoveexcesstissue(i.e.,muscleandfat)fromthebone,whichworks
best by using paper towels and gently rubbing off the tissue.
Figure 1. Isolation of murine femurs and tibiae
(A) Place the mouse face down to the dissecting platform. Cut the skin at the back and expose the legs.
(B) Cut the femur at the hip joint (1.), then take out the whole leg. Separate the femur by cutting the knee joint (2.).
Separate the tibia from the paw (3). (C) Cut off the epiphyses on both sides of the bone. Carefully insert a syringe filled
with DC medium and flush out bone marrow into a 50mL falcon tube.
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STAR Protocols 3, 101664, September 16, 2022 5
Protocol
d. Cut the paw off distal to the ankle, and peel off the remaining skin.
CRITICAL: Avoid to cut the bones, carefully cut behind the epiphyses!
5. Repeat step 4 for the left leg.
6. Spray both legs with 70% (vol/vol) ethanol.
7. Collect femurs and tibiae
a. Working on a 70% (vol/vol) ethanol–soaked towel, hold the distal end of the tibia with a for-
ceps so that the knee joint is pointing away from you and the leg is vertical.
b. Using the tip of slightly opened scissors, strip the calf tissue by pushing it down toward the knee.
c. Grip the tibia and femur with two sets of forceps and gently push against the direction of the
knee joint until the tibia breaks right at the knee.
d. Remove any hanging tissue and place the tibia on the towel.
e. Recover the femur in the same way, snapping the knee off.
8. To remove any remaining flesh from the bones, hold the fleshy end of each piece in a dry paper
towelandpullthebonewithforceps,leavingthefleshinthetowel.Thensqueezetheremaining
fleshy part in the towel and rub it with your fingers (holding it in the towel) to clean the bone off.
9. Collect the bones in a small Petri dish filled with medium on ice.
CRITICAL: If the bone is broken or the soft tissue is inadequately removed, the number of
bone marrow cells harvested may be decreased.
Note: DCmediumcanbestoredat4
C degrees for a maximum of 4 weeks. Check color of
medium before usage. If the color changes from yellowish red to purple-pink (which indicates
pH changes), discard the medium and make fresh one.
10. Disinfect and wash the bones
a. Soak the bones in 70% (vol/vol) ethanol in the first well of the prepared 6-well plate for 1 min.
b. Place them in a second well filled with 2 mL BMDC culture medium (see Materials and Equip-
ment).
c. Move them on to a third well filled with 2 mL BMDC medium.
CRITICAL: Broken bones should not be soaked in ethanol.
11. Collect 10 mL fresh BMDC culture medium in a 10-mL syringe and attach a 27-gauge needle.
12. Remove the tibia from the Petri dish and cut it off at the ankle joint at an angle.
13. Collectthebonemarrow
a. Hold the tibia over a fresh 50-mL tube, with the narrow end of the tibia pointing down. Using
the needle, gently squirt medium onto the tibia.
b. Insert the needle (gently at first) at the top end of the marrow and squirt medium.
c. Pull back the needle and insert it again. Squirt with brief, high pressure pushes.
d. Push the needle in further until the epiphysis has been crossed and medium comes out at the
bottom end of the bone.
e. Continue flushing a couple more times; when the bone is white, discard it (Figure 1C).
14. Repeat steps 12 and 13 for the femur, cutting off at the hip joint just as the tibia was cut at the
ankle joint and using the same 50-mL tube as was used for collection from the tibia.
15. Add an additional 20 mL of BMDC culture medium to the 50-mL tube with a glass pipet and
gently suspend the marrow in the BMDC medium with the pipet.
16. Pass the cell suspension through a 70-mm cell strainer into a new 50-mL tube to remove any re-
maining bone or muscle fragments.
CRITICAL: If performing this step, ensure that the bone marrow is thoroughly suspended
before passing through the cell strainer, otherwise cells will be lost.
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6STAR Protocols 3, 101664, September 16, 2022
Protocol
17. Centrifuge the cell suspension at 250 3gat 22–23C for 5 min to pellet the cells. Discard the
supernatant carefully aspirating it.
18. Lyse the red blood cells (RBC)
a. Add 5 mL / Falcon tube of ice-cold lysis buffer for RBC lysis and gently tap the tube with fin-
gers to mix the lysis solution for 5 min.
b. Add 10 mL of RPMI medium to dilute the buffer after RBC lysis.
19. Centrifuge the cell suspension at 250 3gat 22–23C for 5 min to pellet the cells. Discard the
supernatant.
20. Resuspend the cells in 18 mL of culture medium and mix well. Optionally: count the cells; usually
2–6 310
7
bone marrow cells can be collected from two tibias and two femurs.
21. Day 0: Seed bone marrow cells of one mouse in one 6 well plate, using 3 mL per well with
10–25 ng/mL of GM-CSF in a 37C and humidified 5% CO
2
incubator.
Note: A cell yield of approximately 1 310
7
cells can be expected. In this protocol, the cells of
one mouse were distributed evenlyoverthe6wellsandwerenotcountedinadvance.Cells
can alternatively also be counted and seeded at a density of 1,5 310
6
cells per well.
Dendritic cell culture and creating an atherosclerotic environment
Timing: 6 days
These steps describe how addition of acetylated LDL creates an atherosclerotic environment for the
BMDCs.
22. Day 2, 4, 6: Gently wash cells with pre-warmed PBS and supply them with 3 mL of new, pre-
warmed DC-medium (GM-CSF freshly added 20 ng/mL).
23. To create an atherosclerotic environment, add 10 mg/mL acetylated low density lipoprotein
(acLDL) or control on day 6.
CRITICAL:Alternatively,alsooxidizedLDL(oxLDL)canbeused.Theformationoffoam
cells has been traditionally studied using acLDL (Landers and Lewis, 1993;Jones et al.,
2000). Macrophages that take up acLDL in vitro show phenotypic similarities to foam
cells found in atherosclerotic plaques. However, native LDL, oxLDL, and acLDL are each
trafficked to different endosomes and accumulate in distinct lysosomal compartments,
indicating different endocytic pathways (Wang et al., 2007).
24. Day 7: Collect the dendritic cells as described in the following steps 25–28.
CRITICAL: Be careful to avoid collecting the highly adherent cells (these are considered to
be macrophages) by not pipetting too forcefully) The dendritic cells appear as loosely
adherent ’’heaps’’ on the highly adherent macrophage layer.
Important: Non-BMDCs can be eliminated by early washing steps, discarding highly adherent cells,
and enriching or sorting for CD11c
+
cells (Helftetal.,2015).
Note: If the washings and medium changes are not performed carefully, the survival rate and
number of the BMDCs will be reduced.
Collecting the dendritic cells
Timing: 30 min
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STAR Protocols 3, 101664, September 16, 2022 7
Protocol
This section describes, how the differentiated BMDCs can be harvested for using them in different
assays and analyses.
25. Pre-warm the DC-medium at 37Cinthewaterbath(GM-CSFdoesnothavetobeadded).
26. Remove the medium by aspirating.
27. Collect the cells
a. Pour 2 mL fresh medium over the cells in one well of the 6 well plate with a glass pipette.
b. Pipet up and pour over again, repeat this 2 times and finally transfer the medium containing
theDCsintoa50mLFalcontube.
c. Repeat this with all wellsof the 6 well plate – pool the cells of one plate in one 50 mL Falcon tube.
28. Centrifuge the cell suspension at 250 3gat 22–23C for 5 min to pellet the cells. Discard the
supernatant.
CRITICAL: The optimal culture period to generate BMDC with GMCSF was reported to be
7–8 days (Inaba et al., 1992). We have followed this recommendation and used these cells
for experiments after a maximum of 7 days in culture.
EXPECTED OUTCOMES
CD11c
+
cells accumulate acLDL and secrete ApoE (see Figures 3 and 4). We have shown that the
cells are capable of taking up acLDL by incubating them with increasing concentrations of acLDL
(Figure 3) A quantification of this uptake could be done via detecting LDL with a specific anti-LDL
antibody (for example Anti-LDL antibody GW20089F from Merck) in cell lysates.
CD11c
+
cells are able to perform cholesterol efflux (Figure 5).
The individual protocols are outlined in ‘‘quantification and statistical analysis.’’
QUANTIFICATION AND STATISTICAL ANALYSIS
Flow cytometry
Timing: 2h
Figure 2. Characterization of BM derived cells via FACS after 6 days of cultivation
Loosely adherent BM derived CD11c
+
cells were harvested and incubated with fluorescently labeled antibodies against various surface antigens. 63% of
harvested cells were CD11c
+
MHC-II
+
cells.
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8STAR Protocols 3, 101664, September 16, 2022
Protocol
1. Collect cells as described in the protocol (steps 27–30).
2. Resuspend in FACS buffer at a density of 1 310
6
cells in 50 mL.
3. Incubate with antibodies for 30 min on ice in the dark by adding 3 mL of each antibody. Fill up to
100 mLwithFACSbuffer.
4. Wash with FACS buffer by centrifugation.
5. Fix the cells in 1% PFA for 15 min on ice and wash again afterwards. Unstained cells and cells
treated with unspecific IgG antibodies serve as controls. Fixed cells can be stored at 4Cand
analyzed on the next day.
6. Analyze single cell suspensions on a Flow Cytometer. We used FACSCalibur (BD Biosciences)
with Cellquest Pro Software and a Cytoflex S 4-laser cytometer (Beckman Coulter, Brea, CA,
USA) with Kaluza Analysis 2.1 software (Beckman Coulter). Specific monoclonal antibody binding
is expressed as the geometric mean fluorescence intensity (MFI).
Detection of ApoE in cell culture supernatant via western blot
Timing: 2 days and 3 h
7. Incubate cells on culture day 6 with acLDL for 24 h by adding 10 mg/mL acLDL to the wells con-
taining the cells.
8. Wash cells on day 7 and provide them with serum-free medium for 12 h.
9. Harvest cells like described in the protocol (Steps 25–28). Collect supernatant.
10. Determine protein concentration using commercially available kits (for example PierceBCA
Protein Assay Kit, Thermo Scientific, cat. no. 23225) as described by the manufacturer.
11. Add 25 mLof43Laemmli SDS sample buffer (Bio Rad) to 100 mL sample and boil for 10 min at 95C.
Figure 3. Uptake of acLDL by CD11c
+
bone marrow derived cells
BM derived CD11c
+
cells were loaded with fluorescently labeled acLDL. With increasing acLDL concentrations the fluorescent signal within the cells is
increasing.
Figure4. SecretionofApoEuponuptakeofacLDLbyCD11c
+
bone marrow derived cells
BM derived CD11c
+
cells were loaded with 10 mg/mL acLDL. ApoE was detected in cell culture supernatant via western
blot and quantified using ImageJ. Data are the mean GSD, *P < 0.05.
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STAR Protocols 3, 101664, September 16, 2022 9
Protocol
Note: Samples can be stored at 4C for 12 h after this point.
12. Separate by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels (Invitrogen).
Note: Gelscanbestoredat4
C for 2–3 days wrapped in wet paper before usage.
13. Collect gels, cut off stacking gel and wash gel by putting it shortly in a tray filled with blotting
buffer.
14. Transfer separated protein bands to PVDF-membranes (pre-activated by soaking in methanol
for 1 min) using a semi-dry blotter with 7 V for 1 h.
15. After blotting, block with 5% skim milk in Tris-buffered saline 0.05% Tween-20 for one hour at
22–23C under gentle shaking.
Note: Blocking buffer can be stored at 4Cforaweek.
16. Incubate with primary antibody (Rabbit anti mouse ApoE from Abcam, ab183596) in 5% skim
milk in Tris-buffered saline 0.05% Tween-20 at 4C for 12 h under gentle shaking.
Note: Primary antibody in blocking buffer can be re-used at least 3 times when stored at
20CinaFalcontube.
17. Wash the membrane 3 times for 10 min at RT in Tris-buffered saline 0.05% Tween-20 under
gentle shaking.
Note: The buffer can be stored at 22C–24C for 2–3 weeks.
18. Incubate membrane with secondary antibody (anti rabbit from LI-COR, Bad Homburg,
Germany, diluted 1:15000 in 3% Skim milk in Tris-buffered saline 0.05% Tween-20) for 1 h at
22–23C in the dark under gentle shaking.
Note: Secondary antibody should not be re-used and always made freshly before usage.
19. Wash the membranes 3 times with Tris-buffered saline 0.05% Tween-20 and let dry.
20. Scan with the Odyssey Infrared Imaging System (LI-COR, Bad Homburg, Germany).
Figure 5. Cholesterol efflux analysis of BM derived CD11c
+
cells
revealed that cholesterol efflux was significantly enhanced if
these cells were exposed to an atherosclerotic environment
(loading with acLDL)
Data are the mean GSD, *P < 0.05.
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10 STAR Protocols 3, 101664, September 16, 2022
Protocol
Note: Detection does not have to be performed using LICOR. Chemiluminescence-based
detection with HRP-coupled secondary antibodies works well too.
21. After immunodetection, wash membrane twice with Tris-buffered saline 0.05% Tween-20
and stain with 0.1% Coomassie R-250 (Bio-Rad) in methanol/water, 1:1, for 1 min, destain for
20 min in acetic acid/ethanol/water, 1:5:4, wash with water and let air-dry.
Detection of cholesterol efflux
22. Incubate BM derived CD11c
+
cells with acLDL on day 6 with 10 mg/mL for 24 h.
23. Analyze cells for their ability to perform cholesterol efflux using a cell-based cholesterol efflux
kit by Abcam (ab196985) following the manufacturer‘s instructions. Use murine WT serum as
cholesterol acceptor.
24. Perform read-out on an ELISA-reader according to the manufacturer’s protocol.
LIMITATIONS
In these protocols, we have described several analytic methods using murine BM-derived CD11c
+
cells.
Because we have analyzed similar assays on various cells, we consider these protocols to be beneficial.
However, it has to be kept in mind that BM cell isolation yields a diverse population of cells, which are
not only one distinct cell type but various cells expressing different surface antigens. We characterized
these cells using FACS and found 63% of the harvested cells to be CD11c
+
MHC-II
+
(Figure 2).
TROUBLESHOOTING
Problem 1
Bones are broken during the preparation (step 4) and as a result, high cell yields cannot be achieved.
Potential solution
Initially, explant the whole leg and cut the bones only after you already removed surrounding tissue.
Make sure you cut the bones carefully within the joints.
Problem 2
Cell numbers achieved from bone marrow are very low in step 20.
Potential solution
Flush the bone marrow from both sides until the bone appears white.
Make sure that the bone marrow is really transferred into the tube and is not sticking at the end of the
bone. Do not seed too low cell numbers (in our hands, one 6-well-plate per mouse yielded optimal
cell counts).
Problem 3
FACS analysis shows, that the bone marrow cells are not properly differentiated into BMDCs (see
section ‘‘quantification and statistical analysis’’).
Potential solution
Reduce the number of bone marrow cells seeded onto the culture plate. Extend the culturing period
of the BMDCs. Increase the concentration of GM-CSF. Use a different lot of fetal bovine serum.
Problem 4
Number of harvested cells is very low.
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STAR Protocols 3, 101664, September 16, 2022 11
Protocol
Potential solution
When harvesting the cells, make sure you rinse the cells properly, not too gentle, three times with the
medium before collecting them into the Falcon tube.
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be ful-
filled by the lead contact, Harald F. Langer (harald.langer@uksh.de).
Materials availability
This protocol did not generate unique materials or reagents.
Data and code availability
This protocol did not generate unique materials or reagents.
ACKNOWLEDGMENTS
We thank Jacob von Esebeck, Anke Constantz, and Sarah Gekeler for excellent technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foun-
dation) – project number 374031971 – TRR 240 and the Volkswagen Foundation (Lichtenberg pro-
gram) to H.F.L.
AUTHOR CONTRIBUTIONS
M.S. and R.J.S. performed experiments, analyzed and compiled data, and wrote parts of the manu-
script. H.F.L. conceived the project, analyzed data, and wrote the manuscript.
DECLARATION OF INTERESTS
The authors declare no competing interests.
REFERENCES
Helft, J., Bo
¨ttcher, J., Chakravarty, P., Zelenay,
S., Huotari, J., Schraml, B.U., Goubau, D., and
Reis E Sousa, C. (2015). GM-CSF mouse
bone marrow cultures comprise a
heterogeneous population of CD11c(+)
MHCII(+) macrophages and dendritic cells.
Immunity 42, 1197–1211.
Inaba, K., Inaba, M., Romani, N., Aya, H.,
Deguchi, M., Ikehara, S., Muramatsu, S., and
Steinman, R.M. (1992). Generation of large
numbers of dendritic cells from mouse
bone marrow cultures supplemented
with granulocyte/macrophage colony-
stimulating factor. J. Exp. Med. 176,
1693–1702.
Jones, N.L., Reagan, J.W., and Willingham,
M.C. (2000). The pathogenesis of foam cell
formation. Arterioscler. Thromb. Vasc. Biol. 20,
773–781.
Landers, S.C., and Lewis, J.C. (1993). acLDL binding
and endocytosis by macrophages and
macrophage foam cells in situ. Exp. Mol. Pathol. 59,
38–50.
Sauter, M., Sauter, R.J., Nording, H., Lin, C.,
Olbrich, M., Autenrieth, S., Gleissner, C.,
Thunemann, M., Otero, N., and Lutgens, E. (2021).
Apolipoprotein E derived from CD11c
+
cells
ameliorates atherosclerosis. iScience 25 (1),
103677. https://doi.org/10.1016/j.isci.2021.103677.
Wang, M.D., Kiss, R.S., Franklin, V., Mcbride, H.M.,
Whitman, S.C., and Marcel, Y.L. (2007). Different
cellular traffic of LDL-cholesterol and acetylated
LDL-cholesterol leads to distinct reverse
cholesterol transport pathways. J. Lipid Res. 48,
633–645.
ll
OPEN ACCESS
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