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Chapter 1
Assessment of Toll-Like Receptor Expression and Function by Flow Cytometry
Subhasis Mohanty*,† and Albert C. Shaw*,‡
*Yale School of Medicine, Section of Infectious Diseases,
300 Cedar St., P. O. Box 208022, New Haven, CT 06520 USA
Toll-like Receptors (TLRs) are germline encoded, membrane-associated pattern recognition receptors (PRRs) of the innate immune system that recognize pathogen-associated molecular patterns and couple the innate and adaptive immune responses.1–3 Almost two decades after Janeway put forth the hypothesis of “pattern recognition” and a decade and half after their discovery as PRRs, TLRs have been shown to execute a pivotal role in immune functions of multicellular organisms.4–6 Assessments of TLR function generally requires treatment of cells with defined TLR ligands — such as triacylated lipoproteins recognizing TLR1/2, diacylated lipoproteins recognizing TLR2/6, double-stranded (ds) RNA (TLR3), lipopolysaccharide (TLR4), flagellin (TLR5), single-stranded (ss) RNA (TLR7 or TLR8) and unmethylated CpG-rich DNA (TLR9). Here, we describe considerations for evaluating human TLR function in peripheral blood mononuclear cells (PBMCs) using intracellular cytokine staining to evaluate TLR-induced cytokine production in specific cell lineages.6,7
1. Sample Preparation
Cryopreserved samples of peripheral blood or isolated peripheral blood mononuclear cells (PBMCs) are frequently employed in human immunology studies. The use of such samples has obvious advantages, allowing investigators to analyze large numbers of samples from well-characterized cohorts. In addition, several studies indicate that analyses of B or T cell populations appear to be reasonably stable following freeze-thawing.8,9 However, for other cell types such as monocytes, dendritic cells (DCs), NK cells and especially neutrophils, blood samples should ideally be processed immediately to obtain best results. For example, Toll Like receptor (TLR)-induced cytokine production in monocytes or DCs is affected by both delay in processing and cryopreservation.10 Consequently, we try to standardize all processing procedures, at a minimum providing a maximal window of time for processing and ideally keeping unavoidable delays in sample processing uniform for all subjects.
1.1 Sample collection
• Collect 4 or more 10 mL tubes of blood (BD Vacutainer Sodium Heparin tubes) per subject (expected yield of PBMCs is approximately 1.5–2.0 × 106 per mL of blood).
• Transfer the blood tubes to a tissue culture hood.
• Dilute blood with equal volume of 1× Sterile PBS (Dulbecco’s Phosphate Buffered Saline 1×).
1.2 Gradient centrifugation for isolation of PBMCs
• Layer diluted blood over 15 mL Histopaque (SIGMA 1077) in a 50 mL Polypropylene Conical tube. (Thus 2 × 50 mL tubes for centrifugation per individual)
(Note: Histopaque should be stored at 4°C. It can be filtered through a 0.22 μm Cellulose Acetate filtration unit and 15 mL aliquots stored at 4°C in 50 mL polypropylene conical tubes. However, prior to layering with diluted blood Histopaque must be thawed to room temperature).
• Centrifuge for 20–30 min at 400 × g in a swinging bucket centrifuge without brake at 22°C.
• Carefully transfer the tubes to the tissue culture hood. The PBMCs are in a white ring, the buffy coat, at the interface between Histopaque and medium. Aspirate top layer and stop a few mL above the buffy coat.
• Collect the buffy coat by use of a sterile polyethylene transfer pipette to another 50 mL Polypropylene Conical tube containing 20 mL of cold 1× HBSS (Hanks’ Balanced Salt Solution). Place the tubes on ice/cold until further use.
(Note: HBSS aliquots of 20 mL each can be prepared ahead of time and kept on ice until use).
• Adjust the volume of each tube containing PBMCs to 50 mL with cold 1× HBSS. Centrifuge the tubes at 300 × g at 4°C for 10 min to pellet PBMCs.
• Remove the supernatant and re-suspend the pellets in 50 mL of cold 1× HBSS and place on ice.
• Take a 25 μL aliquot and dilute with 25 μL of trypan blue and obtain cell count using a hemocytometer. Alternatively, cells can be counted using cell counter instruments or a flow cytometer such as the Guava EasyCyte using the ViaCount kit.
• Centrifuge cells at 300 × g at 4°C for 10 m to pellet PBMCs and re-suspend in complete RPMI 1640 medium (with 10% Fetal Bovine Serum and 10U/mL Penicillin/Streptomycin) at 1 × 107/mL and store in ice.
• PBMCs can now be used for downstream applications. For assays of innate immune function, we use freshly isolated PBMCs only.
2. Assessment of Human TLR Function
2.1 TLR Expression on monocytes and DCs
Monocytes express surface TLR1, TLR2, TLR4 and TLR5, and TLR 3 and TLR 8 in endosomes. Similarly, DCs also express TLR1, TLR2, TLR4 and TLR5 on the cell surface and endosomal TLR 3, TLR7/8 and TLR 9. Consequently, endosomal TLRs require assessment via intracellular staining following surface staining for lineage markers. Please refer to table 1 for complete list of antibodies. Each lot of antibodies should be titrated to determine an optimum working concentration.
2.1.1 Surface Staining for TLR Expression
• Dispense 100 μL of the freshly isolated PBMC suspension (a minimum of 1 × 106 PBMCs to analyze DC subsets or at least 0.5 × 106 PBMCs for analysis of monocyte subsets) in each of two wells of a 96-well round bottom tissue culture plate (with one well used for isotype control) per subject.
• Centrifuge at 300 × g for 10 min at 4°C.
• Discard the supernatant by flicking plates upside down and place plates on ice.
• Add 200 μL of ice-cold FACS buffer (1 × PBS containing 0.1% FBS) to each well and re-suspend the pellet.
• Centrifuge cells at 300 x g for 10 min at 4°C.
• Add 100 μL of antibody cocktail (at 2 × final concentration) to respective wells and mix by pipetting using a multichannel pipettor, and incubate plate on ice or 4°C for 20–30 min (For DCs, use Table 1, panels C and D, excluding antibodies recognizing the endosomal TLRs 3, 7, 8 and 9).
• Add 100 μL of 1 × FACS buffer and centrifuge at 300 × g for 10 min at 4°C.
• Remove the unbound antibody by discarding the supernatant by flicking as above.
• Re-suspend pellets i...
