CD14 Antibody, FITC

What is CD14 and why is it a valuable target for FITC-conjugated antibodies?

CD14 is a 50-55 kDa glycosylphosphatidylinositol (GPI)-anchored glycoprotein predominantly expressed on monocytes, macrophages, and at lower levels on granulocytes. It functions as a co-receptor for bacterial lipopolysaccharides (LPS), playing a major role in inflammatory responses to LPS . CD14 acts in concert with LPS-binding protein (LBP) to deliver monomeric lipopolysaccharide to the LY96/TLR4 complex, thereby mediating innate immune responses . Additionally, CD14 serves as a coreceptor for TLR2:TLR6 heterodimer responding to diacylated lipopeptides and for TLR2:TLR1 heterodimer responding to triacylated lipopeptides . The FITC conjugation provides a fluorescent tag with excitation maximum at 495 nm and emission maximum at 519-524 nm, making these antibodies particularly suitable for flow cytometric analysis of monocyte populations .

How do different clones of CD14-FITC antibodies compare in research applications?

Several monoclonal CD14-FITC antibody clones are available for research, each with specific characteristics:

Clone selection should be based on your specific experimental requirements, including species reactivity, application, and whether antagonistic activity would affect your results . For human samples, MEM-15 and 61D3 are well-documented options, while Sa2-8 is better suited for murine studies .

What is the optimal protocol for CD14-FITC staining in flow cytometry?

The general protocol for CD14-FITC staining involves:

• Prepare cell suspension at approximately 1×10^6 cells per 100 μL in PBS with 5% FCS or appropriate buffer .

• Add the recommended amount of CD14-FITC antibody (typically 5 μL of pre-titrated reagent per 10^6 cells or per 100 μL of whole blood) .

• Incubate on ice or at 4°C for 30-45 minutes in the dark .

• Wash cells twice with PBS/FCS buffer.

• Resuspend in appropriate buffer for flow cytometric analysis.

• Use forward scatter versus side scatter plots to identify monocyte populations, then analyze CD14+ cells using appropriate gates .

For pre-titrated antibodies like 61D3, 5 μL (1 μg) per test is recommended for normal human peripheral blood cells . For antibodies requiring optimization, titration experiments should be performed to determine the optimal concentration for your specific sample type .

How should CD14-FITC be incorporated into multicolor flow cytometry panels?

When designing multicolor panels incorporating CD14-FITC:

• Consider spectral overlap: FITC (excitation 495 nm, emission 519-524 nm) may have spillover into PE and other channels, requiring appropriate compensation .

• Use forward scatter versus side scatter gating to identify monocyte populations before analyzing CD14 expression .

• For monocyte subset analysis, combine CD14-FITC with other markers such as CD16, HLA-DR, or CD11b.

• When analyzing peripheral blood samples, acquire at least 30,000 events for robust population identification .

A practical example from the literature demonstrates using CD14-FITC in combination with TLR4-PE and VDR-APC to simultaneously evaluate these markers on monocytes, illustrating the compatibility of these fluorophores in a single panel :

What sample preparation techniques ensure optimal CD14-FITC staining?

For peripheral blood samples:

• Use freshly drawn blood collected in appropriate anticoagulant (EDTA or heparin preferred).

• Process samples within 24 hours of collection for best results.

• If using whole blood: Add 5 μL of pre-titrated CD14-FITC antibody directly to 100 μL of whole blood .

• If using isolated PBMCs: Isolate cells using density gradient centrifugation, wash twice with PBS/FCS, and resuspend at 1×10^6 cells/100 μL before adding antibody .

• For adherent cells (e.g., macrophages): Gentle enzymatic detachment methods are preferred over mechanical scraping to preserve CD14 epitopes.

Proper sample preparation minimizes autofluorescence and non-specific binding while maximizing epitope accessibility. Viability dyes should be included to exclude dead cells, which can bind antibodies non-specifically and compromise data quality.

What controls are essential when working with CD14-FITC antibodies?

Implementing appropriate controls is critical for valid interpretation of CD14-FITC staining:

• Isotype controls: Use isotype-matched FITC-conjugated antibodies (e.g., Mouse IgG2a-FITC for UCHM-1 clone, Mouse IgG2b for some other clones) to assess non-specific binding .

• Unstained controls: Essential for setting background fluorescence levels and determining autofluorescence.

• Single-color controls: Required for compensation when using multiple fluorophores.

• Biological controls:

  • Positive: Samples known to express high CD14 levels (e.g., monocytes)

  • Negative: Lymphocytes or cell lines lacking CD14 expression

For multi-parameter experiments, fluorescence-minus-one (FMO) controls may be valuable for setting accurate gates, especially when analyzing populations with variable CD14 expression.

How can CD14-FITC antibodies be used to differentiate monocyte subsets?

CD14-FITC antibodies can be strategically employed to identify and characterize distinct monocyte subpopulations:

• Classical monocytes: CD14^high^CD16^-

• Intermediate monocytes: CD14^high^CD16^+

• Non-classical monocytes: CD14^low^CD16^high^

For accurate subset identification:

• Use CD14-FITC in combination with CD16 conjugated to a spectrally distinct fluorophore.

• Implement a sequential gating strategy: first identify total monocytes based on FSC/SSC properties, then subdivide based on CD14/CD16 expression patterns.

• Calculate mean fluorescence intensity (MFI) to quantify expression levels rather than relying solely on positive/negative designations .

The experimental design should account for potential variation in CD14 expression under different physiological or pathological conditions, as expression can be altered during activation or differentiation.

What are common issues with CD14-FITC staining and their solutions?

When troubleshooting:

• Verify antibody stability (most CD14-FITC antibodies are stable for one year when stored at 2-8°C protected from light) .

• Check for sodium azide in buffers, which may affect some functional assays while serving as a preservative in storage buffers (typically 0.05-0.09%) .

• Consider clone-specific characteristics, as some have antagonistic activity that might influence functional assays .

What analytical approaches best represent CD14-FITC flow cytometry data?

For robust analysis of CD14-FITC flow cytometry data:

• Gating strategy:

  • Use FSC vs. SSC to identify monocyte populations

  • Create CD14+ gates based on appropriate controls

  • For subset analysis, implement bi-axial plots of CD14 vs. additional markers

• Quantification methods:

  • Percent positive cells relative to isotype controls

  • Mean/Median Fluorescence Intensity (MFI) for expression level quantification

  • Molecules of Equivalent Soluble Fluorochrome (MESF) for standardized comparisons across experiments

• Visualization:

  • Histogram overlays for comparing expression levels between conditions

  • Contour or density plots for visualizing co-expression with other markers

  • Consider transformed scales (biexponential) when populations span wide dynamic ranges

Report both percentage and MFI data, as changes in expression level (MFI) may occur without changes in the proportion of positive cells, particularly in studies of activation or differentiation where CD14 expression may be modulated.

How should CD14-FITC antibodies be stored to maintain optimal performance?

Proper storage is critical for maintaining CD14-FITC antibody performance:

• Store at 2-8°C (not frozen) unless otherwise specified by the manufacturer .

• Protect from light exposure, as FITC is susceptible to photobleaching .

• Avoid repeated freeze-thaw cycles.

• Most formulations include sodium azide (0.05-0.09%) and sometimes BSA (0.5%) as preservatives .

• When properly stored, CD14-FITC antibodies typically remain stable for one year from the date of receipt .

For working dilutions, prepare fresh before use and minimize exposure to light and elevated temperatures. If diluted antibody must be stored, keep at 4°C for short periods only and validate performance before critical experiments.

What quality control measures can verify CD14-FITC antibody performance over time?

To ensure consistent CD14-FITC antibody performance across experiments:

• Include standard samples (e.g., preserved aliquots of characterized PBMCs) in each experiment to track staining intensity over time.

• Monitor both positive staining intensity (MFI) and background (signal-to-noise ratio) to detect deterioration.

• Document lot numbers and maintain a record of performance metrics for each lot.

• For critical applications, consider parallel testing of new and previously verified lots.

• Evaluate sensitivity to detect known biological differences (e.g., LPS-induced changes in CD14 expression).

Implementing these measures helps identify potential antibody degradation before it compromises experimental results and aids in troubleshooting inconsistent findings across experiments.