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Methodology - PowerPoint PPT Presentation

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Methodology. Whole Blood. Total RNA Extraction. RNA Purification and NanoDrop. cDNA Synthesis. Polymerase Chain Reaction (PCR). ConA. PMA. PWM. PHA. OKT3. DEX. Ionomycin. Quantitative PCR. Magnetic Labeling and Magnetic Separation. Flow Cytometric Data Acquisition and Analysis.

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Whole Blood

Total RNA Extraction

RNA Purification and NanoDrop

cDNA Synthesis

Polymerase Chain Reaction (PCR)








Quantitative PCR

Magnetic Labeling and Magnetic Separation

Flow Cytometric Data Acquisition and Analysis


Stimulation Profile

Quantification of IL1F9 Levels by qPCR

Figure 1. PCR product of equal amounts of cDNA amplified with IL1F9 (first row), CD154A (second row), IFN- γ (third row) and IL-10 (fourth row).

Identification of Cell Types Producing IL1F9

Graph 1. qPCR graph results. The x-axis represents the number of amplification cycles required to reach the linear phase. The y-axis represents the delta Rn which displays the magnitude of the signal intensity generated by a given set of PCR conditions.

Figure 2. IL1F9 PCR products following magnetic separation of monocytes. First, third and fifth columns represent the cDNA that only contains monocytes following magnetic separation. The second, fourth and the last columns contains the T and B-cells and other cell types. Columns 1 and 2 are untreated, 3 and 4 are PWM, and 5 and 6 are PHA treated.

Confirmation of MonocyteSeperation

Figure 3. Seperation of monocytes from lymphocytes using MACSQuant analyzer. For the graphs 1 and 2, the x-axis represents forward scatter (FSC-A), and y-axis represents side scatter (SSC-A). For the graphs3 , the x-axis represents FITC-A, and y-axis represents CD14+ cells labeled with PE-A. The graph 3 shows the population of cells that tube 7 (monocytes) contains. This tube includes 90.2% CD14+ monocytes and 3.6% lymphocytes.