May 29, 2026
MonoMacs- From PBMCs to adherent macrophages in culture
This protocol is a draft, published without a DOI.
- 1Washington University in St. Louis
Protocol Citation: Amy Sansone 2026. MonoMacs- From PBMCs to adherent macrophages in culture. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 15, 2026
Last Modified: May 29, 2026
Protocol Integer ID: 317160
Keywords: adherent macrophage, macrophage, macrophages in culture, peripheral blood mononuclear cell, isolating pbmc, pbmc
Abstract
Starting from human blood, this protocol outlines how to isolating PBMCs (peripheral blood mononuclear cells), freeze them down, thaw them, modify them into adherent macrophages, and finally harvest the macrophages to initiate experiments
Protocol materials
suspension culture flask T75Genesee ScientificCatalog #25-214
Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135
Trypsin-EDTA (0.25%), phenol redThermo FisherCatalog #25200114
DPBS, calcium, magnesiumThermo FisherCatalog #14040117
Fetal bovine serum (FBS)BioWestCatalog #S181B-500
Polypropylene Centrifuge Tubes, 50mL Conical bottom tube; Max RCF: 15500xG; High-density polyethylene cap; No rackThermo FisherCatalog #0553868
Trypan BlueInvitrogen - Thermo FisherCatalog #T10282
HEPES (1M)Thermo FisherCatalog #15630130
MEM Non-essential Amino Acid Solution (100)MilliporeSigmaCatalog #M7145
2-Mercaptoethanol Bio-Rad LaboratoriesCatalog #1610710
IMDMThermo ScientificCatalog #12440046
Recombinant Human M-CSF (carrier-free)BioLegendCatalog #574802
Sodium PyruvateGibco - Thermo Fisher ScientificCatalog #11360070
GlutaMAX™ SupplementThermo FisherCatalog #35050079
Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438
Heparin Sodium Injection, USPFresenius KabiCatalog #168718
Penicillin-Streptomycin (10,000 U/mL)Thermo FisherCatalog #15140163
General Use and PrecisionGlide Hypodermic Needles, 18 G x 1.5 in. (38mm); Regular bevel; PinkThermo FisherCatalog #148265D
10 mL BD Luer-Lok SyringeBecton Dickinson (BD)Catalog #302995
Ficoll®-Paque PremiumMilliporeSigmaCatalog #GE17-5442-02
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
Materials and Solutions
DPBS, calcium, magnesiumThermo FisherCatalog #14040117 Fetal bovine serum (FBS)BioWestCatalog #S181B-500 Heparin Sodium Injection, USPFresenius KabiCatalog #168718 Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135 Penicillin-Streptomycin (10,000 U/mL)Thermo FisherCatalog #15140163 Sodium PyruvateGibco - Thermo Fisher ScientificCatalog #11360070 GlutaMAX™ SupplementThermo FisherCatalog #35050079 HEPES (1M)Thermo FisherCatalog #15630130 MEM Non-essential Amino Acid Solution (100)MilliporeSigmaCatalog #M7145 2-Mercaptoethanol Bio-Rad LaboratoriesCatalog #1610710 IMDMThermo ScientificCatalog #12440046 Dimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2438 Recombinant Human M-CSF (carrier-free)BioLegendCatalog #574802
| A | |
| LRS Buffer: | |
| 1L PBS | |
| 20 ml FBS | |
| 100ul Heparin (from DCM pharmacy, [email protected]) (10K U/mL 5mL stock.) | |
| Per the protocol below, 100uL heparin into 1L, with 1U/mL final conc. |
| A | |
| PBMC Thawing Media | |
| For 200ml: | |
| RPMI 170ml | |
| FBS 40ml | |
| Pen/Strep 2ml | |
| Na pyruvate 2ml | |
| Glutamine (GlutamaX) 2ml | |
| Hepes buffer 2ml | |
| MEM NEAA 2ml | |
| Our stock BME is 14.2M (BioRad) = 3.87ul BME + 1ml water = 55mM BME | |
| β- mercaptoethanol (stock 55 mM) 0.363ml | |
| Sterile filter the buffer. Store at 4oC |
| A | |
| PBMC Freezing Media | |
| For 50ml | |
| -25ml IMDM | |
| -5ml DMSO | |
| -20ml FBS |
Human Monocyte Macs media:
Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135 = 44.4ml
GlutaMAX™ SupplementThermo FisherCatalog #35050079 = 500ul
Recombinant Human M-CSF (carrier-free)BioLegendCatalog #574802 50 ng/ml M-CSF = 50ul
Fetal bovine serum (FBS)BioWestCatalog #S181B-500 FBS = 5 ml
R10 media
Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135 plus
10% Fetal bovine serum (FBS)BioWestCatalog #S181B-500
Harvest PBMCs from LRS chambers:
Polypropylene Centrifuge Tubes, 50mL Conical bottom tube; Max RCF: 15500xG; High-density polyethylene cap; No rackThermo FisherCatalog #0553868 General Use and PrecisionGlide Hypodermic Needles, 18 G x 1.5 in. (38mm); Regular bevel; PinkThermo FisherCatalog #148265D 10 mL BD Luer-Lok SyringeBecton Dickinson (BD)Catalog #302995
DPBS, calcium, magnesiumThermo FisherCatalog #14040117
Ficoll®-Paque PremiumMilliporeSigmaCatalog #GE17-5442-02
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
| A | |
| For each chamber: | |
| 10ml syringe | |
| 18 gauge needle | |
| 2 x 50ml conical, labeled with sample ID | |
| -Put 20ml LRS buffer into one of the conicals | |
| -cut off chamber tube on skinny side, close to top | |
| -cut chamber tube on fat side, put fat side onto conical | |
| -put 18 gauge needle on 10ml syringe and fill with air | |
| -flush chamber with air | |
| -fill syringe with 10ml LRS buffer (from the other conical) | |
| -flush the chamber with buffer | |
| -repeat flush with the remaining 10ml buffer from the conical | |
| -fill syringe with air and flush the chamber with air | |
| -add buffer to 40ml total and pipet up and down to mix | |
| -Divide the 40ml of sample into two 50 ml conicals (20ml each) | |
| -add PBS to total volume 30ml in each conical | |
| -underlay 12 ml Ficoll-Paque Plus (draw up 12 ml ficoll, put the serological pipet | |
| at the bottom of the conical and release the pipet from the pipet gun) | |
| 400 rcf 30min RT no brake | |
| -remove the top, largest, pinkish-yellow, clear layer of plasma carefully | |
| -transfer the second, narrower, cloudy, white layer (PBMC) into a 50ml conical | |
| -(the bottom two layers are Ficoll (white/clear) and RBCs, etc. (dark red) | |
| -if collected layer is more than 25ml, split into two conicals | |
| -bring volume up to 50ml using PBS | |
| -spin down at 1300 rpm, 4 min. at RT | |
| -aspirate supernatant | |
| -add 3-5 ml ACK lysing buffer, depending on pellet size | |
| -incubate 2-3 min. at RT, gently mix the pellet by flicking the tube gently! | |
| -quench with 30-45ml PBS | |
| -spin down at 1300 rpm, 4 min. at RT | |
| -aspirate supernatant | |
| -resuspend pellet in PBMC thawing media, 25ml | |
| -count cells | |
| -seed desired amount in suspension flask, T175 | |
| -freeze down 24-25 tubes in PBMC Freezing media |
To modify the PBMCs into adherent macrophages:
2d
Spin down 25 mL (12.5 mL PBMC suspension in two 15 mL conicals) at1300 rpm, 20°C, 00:04:00 (4min) Resuspend each pellet in10 mL
Transfer 10 mL of monocyte suspension into each of two suspension culture flask T75Genesee ScientificCatalog #25-214
Incubate cultures in a37 °C cell culture incubator
Equipment
Heracell™ 240i CO2 Incubator, 240L
NAME
CO2 Incubator
TYPE
Thermo Scientific™
BRAND
51032875
SKU
LINK
with 5% CO2 / 95% air for 48 hr.
Rinse the cultures 3 times with 10 mL Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135 (pre-warmed to 37 °C), gently removing the culture medium to avoid dislodging any loosely attached cells.
Following rinsing, add fresh Human Monocyte Macs media, changing media every 48:00:00 until monocytes differentiate and proliferate sufficiently to become confluent.
2d
Harvesting Macrophages to Initiate Experiments
- Rinse differentiated macrophages in flask with 10 mL pre-warmed 37 °C DPBS, calcium, magnesiumThermo FisherCatalog #14040117 Dulbecco’s phosphate-buffered saline without Ca2+ and Mg2+ (DPBS) before adding 3 mL pre-warmed 37 °C Trypsin-EDTA (0.25%), phenol redThermo FisherCatalog #25200114
Place flask in cell culture incubator for 00:10:00 (5-10 min) at which time approximately 90% of the macrophages should have rounded and detached. Verify this by microscopic examination of the flask.
10m
Add5 mL of Gibco™ RPMI 1640 MediumThermo Fisher ScientificCatalog #11875135 containing 10%Fetal bovine serum (FBS)BioWestCatalog #S181B-500 to stop trypsinization.
Transfer the macrophage cell suspension from the flask into a Polypropylene Centrifuge Tubes, 50mL Conical bottom tube; Max RCF: 15500xG; High-density polyethylene cap; No rackThermo FisherCatalog #0553868 300 x g, 20°C, 00:05:00 , (5 min) , and discard the supernatant.
5m
Gently resuspend the macrophages into 5 mL of Human Monocyte Macs media
Mix 10 µL of macrophage cell suspension with 10 µL Trypan BlueInvitrogen - Thermo FisherCatalog #T10282 solution and determine macrophage cell number and viability (typically > 95%) using
Equipment
Countess II FL
NAME
Cell Counter
TYPE
Invitrogen
BRAND
AMQAF1000
SKU
LINK
Based on the cell count, add additional Human Monocyte Macs media to achieve the desired seeding cell density.
Protocol for Thawing PBMCs:
Warm PBMC Thawing Media to 37 °C in a water bath before beginning the thawing procedure.
Remove the vial(s) from liquid nitrogen (LN2 Box C09) and wipe off the outside of the vial, and spray with 70% ethanol.
Place the vial(s) in cell culture incubator at 37 °C for 00:10:00 at least 10 min
10m
Remove from incubator, spray with 70% ethanol, and wipe off the outside of the vial.
Transfer the clean, dry vial to the biosafety hood.
Gently pipette warmed PBMCs into tube with warmed PBMC Thawing Media . The final volume should be ≥ twice the volume of the cell suspension.2 mL depending on volume of cells, usually 1 ml cells in cryovial
330 rcf, 20°C, 00:10:00 , 10 min
10m
Pour off supernatant.
Gently flick the tube to break up pellet. DO NOT pipet up and down or vortex!!
Repeat wash.
330 rcf, 20°C, 00:10:00 , 10 min
10m
Pour off supernatant.
Re-suspend cells in warm PBMC Thawing Media
Determine the cell number and viability with Trypan BlueInvitrogen - Thermo FisherCatalog #T10282
Equipment
Countess II FL
NAME
Cell Counter
TYPE
Invitrogen
BRAND
AMQAF1000
SKU
LINK
Protocol references
S. Neron et al. "Characterization of Mononuclear cells remaining in the LRS chamber..."Transfusion Vol. 47, issue 6, pg. 1042
S. Neron et al. "Whole Blood LRS..." Transfusion, Vol. 46, issue 4 pg. 537 https://pubmed.ncbi.nlm.nih.gov/30189061/
Am J Respir Cell Mol Biol, 2022 Jan;66(1):64-75. doi: 10.1165/rcmb.2021-0190OC.
Murine Ex Vivo Cultured Alveolar Macrophages Provide a Novel Tool to Study Tissue-Resident Macrophage Behavior and Function
J Vis Exp, 2016 Jun 30:(112):54244. doi: 10.3791/54244.
Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages
Immunity Volume 47, Issue 5, 21 November 2017, Pages 903-912.e4
The Cytokine TGF-β Promotes the Development and Homeostasis of Alveolar Macrophages
Xueyang Yu 1, Anne Buttgereit 1, Iva Lelios 1, Sebastian G. Utz 1, Dilay Cansever 1, Burkhard Becher 1, Melanie Greter 1 2https://doi.org/10.1016/j.immuni.2017.10.007
Acknowledgements
Procedure adapted from Fehniger Lab, MBE's protocol and JVW, Stallings Lab