A RAPID AND SIMPLE MICROFLUOROMETRIC PHAGOCYTOSIS ASSAY
A RAPID AND SIMPLE MICROFLUOROMETRIC PHAGOCYTOSIS ASSAY
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DOI：
10.1016/0022-1759(93)90400-2
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发表时间：
1993-06-04
影响因子：
2.2
通讯作者：
LAU, BHS
中科院分区：
文献类型：
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作者：
WAN, CP;PARK, CS;LAU, BHS
用荧光素标记的大肠杆菌K-12颗粒建立了一种研究吞噬作用的显微荧光法。该技术基于荧光颗粒的摄取和测定结束时细胞外荧光的淬灭。鼠巨噬细胞系J774用作吞噬细胞模型。从组织培养瓶中收获细胞，并调整至1 × 10（6）个细胞/ml。然后将它们分配到96孔组织培养板中，100穆尔/孔，并在37 ℃、5%CO2下孵育1小时，以使细胞粘附到威尔斯孔的底部。吸出培养基并加入100穆尔。加入悬浮在Hanks缓冲液中的大肠杆菌颗粒。将板进一步孵育不同的时间段。通过抽吸除去威尔斯孔中的缓冲液。然后通过加入100 μ l台盼蓝（250 μ g/ml，pH4.4）淬灭细胞外荧光。1分钟后去除染料。使用计算机化微孔板荧光计在485 nm激发和530 nm发射下直接测量威尔斯孔中与细胞内荧光颗粒相关的荧光强度。该测定提供了吞噬活性的快速和客观的测量。使用培养的细胞系和96孔微量滴定板格式，该测定可以促进筛选大量的各种生物和药理学物质对吞噬作用的调节作用。
A microfluorometric method for phagocytosis study has been developed using fluorescein conjugated Escherichia coli K-12 particles. This technique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay. A murine macrophage cell line, J774, was used as a phagocyte model. The cells were harvested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml. They were then dispensed into a 96-well tissue culture plate, 100 mul/well, and incubated at 37-degrees-C in 5% CO2 for 1 h to allow cells to adhere to the bottom of the wells. The culture medium was aspirated and 100 mul of fluorescent E. coli particles suspended in Hanks' buffer were added. The plates were further incubated for various time periods. Buffer solution in the wells was removed by aspiration. Extracellular fluorescence was then quenched by adding 100 mul of trypan blue (250 mug/ml, pH 4.4). The dye was removed after 1 min. The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. This assay provided a rapid and objective measurement of phagocytosis activity. Using a cultured cell line and a 96-well microtiter plate format, this assay can facilitate the screening of a large number of various biological and pharmacological substances for their modulating effects on phagocytosis.