This document describes standard research protocols used for generation of human monocyte-derived macrophages and DC in the Hutchinson work-group.
Method Article
Standard Protocols for Generation of Monocyte-derived Cell Types
https://doi.org/10.1038/protex.2018.065
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posted 11 Jul, 2018
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This document describes standard research protocols used for generation of human monocyte-derived macrophages and DC in the Hutchinson work-group.
This document describes standard research protocols used for generation of human monocyte-derived macrophages and DC in the Hutchinson work-group.
Abbreviations
Mreg: Regulatory macrophage
DPBS: Dulbecco’s modified phosphate buffered saline
M-CSF: Macrophage colony-stimulating factor
FBS: Fetal bovine serum
HABS: Human AB serum
IFN-γ: Interferon-gamma
LFH: Laminar flow hood
PR: Phenol Red
CPDA: Citrate-Phosphate-Dextrose-Adenine solution
LRS: Leucocyte Reduction System
General reagents
RPMI1640 without phenol red (BE12918F, Lonza-Biozym)
Glutamax100X (35050038, Gibco-life)
Pen-Strep 100X (15140-122, Gibco-life)
rhM-CSF (216-MC-025, R&D)
rhIFN-γ (IF002, Millipore)
CD14 microbeads (130-050-201, Miltenyi)
Albunorm 20% - Human Albumin (Octapharma)
Citrate-phosphate-dextrose-adenine solution (C4431, Sigma)
Dulbecco’s Phosphate Buffered Saline without Ca2+ or Mg2+ (D8537, Sigma)
Biocoll Separating Solution (L6115, Millipore-Biochrom)
Human AB Serum (ZKT Tübingen)
FBS (10270, Life/Gibco)
X-VIVO 10 (BE04-380Q, Lonza)
hrGM-CSF (300-03, Peprotech)
hrTNF-α (300-01A, Peprotech)
hrIL-4 (200-04, Peprotech)
LPS (L2018, Sigma)
Dexamethasone (D4902, Sigma)
Materials
Stericup+ Steritop 250ml (SCGPU02RE, Millipore)
QuadroMACS Separation Unit (130-090-976, Miltenyi)
Pre-separation Filters (130-041-407, Miltenyi)
LS Columns (130-042-401, Miltenyi)
Sarstedt Cell+ 6-well plates (833.920.300, Sarstedt)
1. PREPARATION OF REAGENTS
1.1 Preparation and storage of rhM-CSF
Store at -20°C for up to 8 weeks.
1.2 Preparation and storage of rhIFN-γ
Store at -20°C for up to 8 weeks.
1.3 Heat inactivation and storage of human AB serum and FBS
Store aliquots at -20°C for up to 4 weeks.
1.4 Preparation of MACS buffer
CPDA is used in preference to EDTA. Human albumin is used in preference to bovine albumin.
Filter mixture. Store at 4°C.
1.5 Preparation of Mreg culture medium
Protocol to produce 100 ml medium. Scale as required.
Filter medium mixture. Medium must be used fresh.
1.6 Preparation of FBS-containing culture medium
Protocol to produce 100 ml medium. Scale as required.
Filter medium mixture. Medium must be used fresh.
2. STARTING MATERIAL
2.1 Obtaining apheresate from LRS chamber
Perform flow cytometry analyses (eg. CD14+ monocytes).
2.2 Separation of PBMC by density gradient centrifugation
Ficoll density gradient centrifugation is used to enrich PBMC prior to CD14 selection by MACS. This is necessary when working with apheresates from LRS chambers, buffy coats or whole blood, owing to their high content of neutrophils (which may be CD14low) and erythrocytes.
Transfer required volume of cell suspension to a 50 ml Falcon tube.
3. MREG CULTURE
3.1 Day_0 of culture: Isolation of monocytes (~90 minutes)
Take aliquots of cells for (i) Trypan Blue count and (ii) CD14 analysis by flow cytometry.
3.2 Day_0 of culture: Seeding of Mreg cultures: ~30 min
Place plates into 37°C 5%CO2 incubator on horizontal shelves.
3.3 Day_6 of culture: IFN-γ stimulation: ~30 min
Replace plates into incubator and incubate for a further 18 to 24 hours.
3.4 Day_7 of culture: Cell harvest: ~40 min
b. With experience it is possible to judge cell density. Look also for cell debris suggesting death of cells in culture.
Assess cell phenotype by flow cytometry.
4. GENERATION OF OTHER MONOCYTE-DERIVED CELL TYPES
4.1 Generation of monocyte-derived dendritic cells
Assess cell phenotype by flow cytometry.
4.2 Generation of polarised macrophages
recipients. J Immunol. 1;187(5):2072-8 (2011)
The authors declare no competing financial interest.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 11 Jul, 2018
You are reading this latest protocol version
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