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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

     2.1 mouse macrophage FITC-quantitative observation of

    Escherichia coli

     By Escherichia coli Gram stain images (Figure 1) can be seen: Escherichia coli as a Gram-negative bacteria, and no

    other bacterial contamination, can be used in phagocytosis experiments; from Escherichia coli fluorescence microscope observed (Figure 2) can be seen: The carbonate buffer (pH = 9) Preparation of fluorescein (FITC) can be effectively marked Escherichia coli; at different time periods G W staining of

    macrophage phagocytosis of Escherichia coli Bacteria process the image (Figure 3) can be seen: In the 0 ~ 20min macrophages gradually increasing the amount of E. coli phagocytosis (macrophage foam to increase the amount of E. coli), 30min or so, when giant The maximum amount of macrophage phagocytosis, 40min after the macrophages have reduced the amount of trend

    (phagocytic vacuole obvious bacteriolytic phenomenon, bubbles form inside the E. coli gradually blur).

     2.2 mouse macrophage FITC-kinetic characteristics of

    Escherichia coli

     Mouse macrophages and FITC-Escherichia coli at 37 ? for

    different time after the actual macrophage phagocytosis rate 0,10,20,30,40,50 min, respectively for 0%, 11.20 ? 2.0%,

    21.57 ? 2.1%, 62.22 ? 5.0%, 54.58 ? 3.5%, 37.22 ? 4.1%, at

    30min or so that the peak (Table 1, Figure 4). Table 1 macrophage phagocytosis of Escherichia coli of the kinetic data (omitted)

     3 Discussion

     Immune function in human cells is generally believed that should include white blood cells, red blood cells and macrophages in the three major systems, the corresponding

    detection indicators are as follows: in order to CD4 and CD8-

    based T-cell subsets in order to RBC C3bRR and RBC ICR

    for the Lord's red blood cell immune function tests and in the

    rate of macrophage-based detection of macrophage phagocytosis. However, in the clinical detection method due to cellular immune function in a more complicated, often for the first two tests, as well as for some autoimmune diseases, and immune dysfunction related to disease diagnosis and treatment, comprehensive assessment of the immune status of paramount

    importance.

     Detection of MP function is one of the most common way to detect their phagocytic rate of bacteria, the traditional method is to take peripheral blood smears, after mixing with the bacterial staining, observed under the microscope MP

    phagocytic rate of bacteria. This method is generally to find more than 200 the number of MP in the eating bacteria MP, operating more cumbersome, and the form of small, under a microscope to observe the phagocytic rate of bacteria more

    difficult. Therefore, researchers have reported that neutrophils measured by flow cytometry, fluorescent-labeled

    cells in the phagocytosis of bacteria [4,5], but the macrophage function of test-reported.

     In this study, on the basis of previous research [6],

    choose the FITC and EB double fluorescent labeling substance detection of MP against Escherichia coli phagocytosis. The detection principle is: first, the use of FITC in carbonate buffer is marked E. coli, and then labeled MP phagocytosis of

    Escherichia coli, phagocytosis is completed, adding EB dye-MP,

    at the 488nm laser in the large intestine under the MP coli bacteria on the first issue of the green FITC fluorescence, its fluorescence can also stimulate MP on the EB dye to produce red fluorescence, red fluorescence by calculating the expression ratio reflects the MP for the phagocytosis of Escherichia coli. The results showed that: 0 ~ 20min in the rate of phagocytosis increased gradually; 30min phagocytic rate of about platforms, with the consistency of the

    information; 40min later, however, reduced the rate of phagocytosis, it is possible through the role of MP in the MP bacterial lysozyme digestion, decomposition, release into the cells, leading to bacterial reduction of MP, fluorescence

    diminished capacity. Of these, flow cytometry histogram of fluorescence intensity of the CV averaged 3.9%, we can see, automatic flow cytometry used to detect macrophage function, not only the accuracy of measurements, reproducible and easy to operate , fast, and the cell membrane to maintain the

    integrity of MP is able to directly reflect the MP phagocytosis of bacteria, while made up for difficult to distinguish between weak FITC fluorescence to detect weaknesses.

     Our results fully proved by flow cytometry of FITC EB

    method can be simple, rapid and reproducible quantitative detection of macrophages in cell phagocytosis of Escherichia coli.

     References

     1 Zhang Ying-hua, Yin Ying, Zhang Li, et al. Flow cytometry macrophage ratio method and its

    application. .2002,23 Journal of Fourth Military Medical University (17): 1559 ~ 1561.

     2 White Owen C, Alexander JW, Sramkoski RM, et al.Rapid whole blood microassay using flow cytometry for measuring neutrophil phagocytosis. J Clin Microbiol, 1992,30

    (8): 2071 ~ 2076.

     3 Zhao Xiu-chun, Yao Chunyan, LI Bai-qing, et al. Flow

    cytometry neutrophil phagocytosis in mice a methodological approach. Basic Medical .2004,29 (5): 388 ~ 390.

     4 Vander Top EA, Perry GA, Gentry Nielsen MJ. A novel

    flow cytometric assay for measurement of in vivo pulmonary neutrophil phagocytosis. BMC Microbiology. 2006,6:61.

     5 Li W, Chung SC. Flow cytometric evaluation of leukocyte function in rat whole blood. In Vitro Cell Dev Biol Anim.

    2003,39 (10): 413 ~ 419.

     6 Bassoe CF.Flow cytometric quantification of

    phagocytosis in acute myeloid leukemia.Acta Haematol.1999, 102 (4): 163 ~ 71. Reposted elsewhere in the paper for free download http://

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