4 Notes

1. Prior to the assay, it is important to determine the optimal ratio of cell/beads for optimal results. Addition of excess beads may lead to unwanted “background noise,” whereas an insufficient number of beads may lower the signifi cance of the fi ndings. We recommend for microglia a cell/beads ratio of 1:100.
2. Of course, other plates, i.e., 12- or 24-well plates, can be used as well. However, then lower density seeding is required. Cell numbers may then be insuffi cient to record an appropriate number of life events.
3. Regardless of the uptake mechanism, it is considered plausible that polystyrene beads spend some time on the exterior of the cell membrane prior to translocation to the intracellular environment. To distinguish between uptake and adsorption, other methods can be used as well:
   (a) “Quenching” of adsorbed fl uorescent markers with agents like ammonium acetate or trypan blue was proposed [ 18 , 19 ].
   Due to overlapping, further discrimination of peaks is not possible. However, since the first peak is also detectable at 4 °C, it may just result from beads attached to the cell surface. Thus, the MFI at 37 °C minus the MFI at 4 °C is the amount of incorporated fl uorescent latex particles phagocytosed by a given number of microglia Phagocytosis Assay for Microglia (b) Phagocytosis but not adsorption of the beads to the cells can be completely abolished by cytochalasin D (5 μg/ml), an inhibitor of actin polymerization [ 13 ]. The use of cytochalasin
   D is a very elegant approach to correct for the fluorescence/number of the adsorbed beads.
   We recommend the use of confocal microscopy for a qualitative distinction between beads taken up by cells or merely attached to the plasma membrane ([ 13 ], Fig. 3 ). It should be noted that fluorescent latex beads may be phagocytosed in a different way and at a different rate than other particle types. Unfortunately, little work has been carried out on exploiting the mechanism of the intracellular uptake of polystyrene latex beads. To our knowledge, there is only one report proposing that CD11c may be involved in the
   uptake of non-opsonized polystyrene beads [ 20 ].
4. For better results it is important to maintain the cells at 4 °C for further steps. All solutions used and the centrifuge have to be precooled and maintained at 4 °C.
5. The number of washes is suffi cient to remove unattached beads since further washing did not reduce the adsorption-corrected fluorescence. However, thorough washing is important to
   eliminate all the non-phagocytosed beads that are either fl oating in the medium or attached to the cell surface. Residual beads can infl uence the fi nal result as they can be detected on the flow cytometer and lead to false signals.
6. Vigourous pipetting can result in death of few cells. The dead cells can be excluded from the analysis by using propidium iodide or any other viability tracking dyes (e.g., 7-AAD).
   We strongly recommend to perform the phagocytosis assay Fig. 3 Representative z-stack series of confocal microscopic images of two microglia cells ( red , labelled with anti-OX42) defi nitely demonstrate that the particles were completely internalized and not merely attached to
   the outer membrane Refi k Pul et al.

译文

1. 在检测之前，确定细胞/珠粒的最佳比例以获得最佳结果是很重要的。添加过多的珠子可能会导致不必要的“背景噪声”，而珠子数量不足可能会降低发现的显著性。我们建议小胶质细胞/珠粒的比例为1:100。
2. 当然，其他板，即12或24孔板，也可以使用。然而，低密度播种是必需的。细胞数量可能不足以记录适当数量的生命事件。
3. 不管吸收机制如何，人们认为聚苯乙烯珠在转位到细胞内环境之前在细胞膜外部花了一段时间是合理的。为了区分吸收和吸附，还可以采用其他方法:
   (a)提出用醋酸铵或台锥蓝等药剂对吸附的荧光标记物进行“猝灭”[18,19]。由于重叠，进一步区分峰是不可能的。然而，由于第一个峰在4°C也可以检测到，它可能只是由附着在细胞表面的珠子引起的。因此，37°C时的MFI减去4°C时的MFI是被给定数量的小胶质细胞吞噬的荧光乳胶颗粒的掺入量。
   小胶质细胞吞噬实验
   (b)细胞松弛素D (5 μg/ml)，一种肌动蛋白聚合抑制剂，，可以完全消除吞噬作用，但不吸附小胶质细胞。使用细胞松弛素D是一种非常优雅的方法来校正吸附珠的荧光/数量。我们建议使用共聚焦显微镜来定性区分被细胞吸收的珠粒或仅仅附着在质膜上的珠粒([13]，图3)。应该注意的是，荧光乳胶珠可能以不同的方式和不同的吞噬速率比其他颗粒类型。不幸的是，关于聚苯乙烯乳胶珠细胞内摄取机制的研究很少。
   据我们所知，只有一份报告提出CD11c可能参与了非调理聚苯乙烯珠[20]的摄取。
4. 为了获得更好的结果，重要的是将细胞保持在4°C，以便进行进一步的步骤。所有使用的溶液和离心机必须预冷却并保持在4°C。
5. 洗涤次数足以去除未附着的珠粒，因为进一步的洗涤不会降低吸附校正的荧光。然而，彻底的清洗对于清除漂浮在培养基中或附着在细胞表面的所有未被吞噬的珠子是重要的。残留的珠粒会影响最终结果，因为它们可以在流式细胞仪上检测到，并导致假信号。
6. 6剧烈的移液可导致少数细胞死亡。使用碘化丙啶或任何其他活性追踪染料(如7-AAD)可将死亡细胞从分析中排除。图3两个小胶质细胞的代表性z-stack系列共聚焦显微图像(红色，标记有抗ox42)明确表明，颗粒完全内化，而不仅仅是附着在外膜上。