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By flow cytometry macrophage phagocytosis of Methods_3256

By Kristen Stone,2014-10-30 11:43
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By flow cytometry macrophage phagocytosis of Methods_3256

By flow cytometry macrophage phagocytosis of Methods

     Abstract Objective: To establish detected by flow

    cytometry using mouse peritoneal macrophages induced by phagocytosis of methods. Methods: Screening of adhesion of activated macrophages, and then with the carbonate buffer prepared with fluorescein (FITC) labeled Escherichia coli mixture, 37 ? incubation, each taking a small amount of fixed 10min after being stained giant EB macrophage nucleus, using flow cytometry to calculate macrophage phagocytosis rate for

    Escherichia coli. Results: FITC can be effectively marked Escherichia coli; mouse peritoneal macrophages induced by E. coli can be swallowed, and reached the maximum peak 30min. Conclusion: Flow cytometry detection of murine macrophage

    phagocytosis of Escherichia coli is to determine the FITC-

    macrophage function is simple, rapid and reproducible quantitative method.

     Key words macrophage phagocytosis of Escherichia coli by flow cytometry

     Establish a New Method of Detecting Phagocytosis of

    Macrophage by Flow Cytometry

     Abstract Objective: To use a new method of detecting phagocytosis of macrophage induced from the Belly of mouse by flow cytometry. Methods: Firstly, the living macrophages were collect by cultivating and adherencing, and bacterium coli (E. coli) was labeled FITC. Secondly , E. coli was mixed with macrophages, every 10 minute to take out of small amounts of mixed liquor, and add a little of EB dye. Finally, the fluorescent intensity was measured by flow cytometry. Results:

    The E. coli was labeled well by FITC in carbonate buffer. Macrophage can phagocytize E. coli and the efficient rat of

    phagocytosis was highest about at 30 minute. Conclusion: The method that detecting phagocytosis of macrophage phagocytizs

    E. coli is convenient, fast, good repeatability.

     Key words macrophage; phagocytosis; bacterium coli; flow cytometry

     Macrophages (macrophage, MP) is the body's main immune cells of natural immunity, phagocytic rate was a direct reflection of macrophage phagocytosis. Determination of the rate of phagocytosis is usually the method is to engulf bacteria MP staining was observed under a microscope, counting methods, there are researchers using flow cytometry (Flow Cytometry, FCM) detection of MP phagocytosis [1,2]. We have detected MP eating bacteria experiments in mice found that mice form a smaller MP, staining was observed under the microscope more difficult to calculate the rate of phagocytosis. This study tests the establishment of flow

    cytometry to detect FITC EB phagocytic function of MP in

    order to find a simple, objective and reproducible method.

     1 Materials and methods

     1.1 Experimental Materials

     1.1.1 Main reagents

     FITC; EB; 2% starch broth; Escherichia coli DH5A; 2% agar

    LB solid medium; containing serum-free medium; four-color flow

    cytometry calibration microspheres (CaliBRITEBeads, U.S. BD company).

     1.1.2 Experimental Animal

     BALB / C mice, 25, body weight (19 ? 2) g, by the

    hospital to provide Immunology saved.

     1.1.3 Main equipment and software

     Watertight constant temperature incubator, model: GNP

    9270 (Experimental Equipment Co., Ltd. Shanghai Jingyi Wang); self-control CO2 cells incubated box, Model: 2300 (U.S. Shellab companies); spectrophotometer, model: Jenway 6305 (Barloworld companies); Zeiss fluorescence microscope, model:

    ZEISS AXIO Imager.A1 (Carl Zeiss optical instrument (Shanghai) International Trading Co., Ltd.); flow cytometry, Model: FACSCalibur (U.S. BD Inc.); CellQuest software.

     1.2 Experimental Methods

     1.2.1 E. coli culture and marker fluorescein (FITC)

     Inoculated E. coli in LB solid medium 2% agar surface, Min, in the 37 ? constant temperature incubator impermeable incubated 48h, take a small amount of colonies washed with PBS, gram (Figure 1) and G W staining (Figure 3 ), and

    adjust the concentration (OD ? 0.40 at 620nm Office).

    Fluorescein-labeled E. coli [3]: Centrifugal to the

    supernatant, add 250ul 1% Triton X 100 and 1ml FITC (0.01mg

    FITC/500ul carbonate buffer) 37 ? dark incubated 15min,

    centrifugation to the supernatant, a small amount of PBS re-

    suspended bacteria stand-by, and used fluorescence microscopy (Figure 2).

     1.2.2 macrophage phagocytosis of E. coli

     Induction, screening peritoneal macrophages: the groin into the 1ml2% starch broth liquid, 48h after the open peritoneal exudate collection, medium wash, into the culture bottle 37 ? CO2 incubation boxes were incubated 2h, to collect adherent cells (1 × 107event) in the centrifuge tube 4ml medium re-suspended stand-by, and G W staining (Figure

    3). Detection of macrophage phagocytosis of E. coli: E. coli-

    FITC added in the macrophage culture medium at 37 ? the level

    of shaking bed (slow rock), every 10min to take shaken liquid 0.5ml, PBS wash, 70% ethanol Fixed 10min, after centrifugation by adding 1ml EB (0.5μg/ml), 37 ? dark incubation 15min

    under test. The first use of BD CaliBRITE Beads reagents to adjust equipment to set the basic parameters of instrument, the sample after the first use FSC H / SSC H delineation

    of MP, and then collect the red fluorescence intensity, and

    calculate the actual phagocytic rate (a certain moment a certain moment of the actual phagocytic rate = phagocytic rate-0min when the phagocytic rate), while taking a small amount of G W staining (Figure 3).

     1.3 Statistical Analysis

     SPSS11.0 software used for statistical analysis, data to ? s indicated, P <0.05 as statistically significant. Reposted elsewhere in the paper for free download http://

     2 Results