Notes

©1 Other homogenization media may be permissible and may need modulating if other cell types are used; see ref. 35 for more details.

©2 The device of choice is the ballbearing homogenizer. Sometimes a syringe needle with a lower gauge number (wider orifice) will work satisfactorily. As with the homoge-nization medium, the homogenization protocol will need to be tailored to the cell type. See ref. 35 for more information.

© Either the Beckman Fraction Recovery or the Labconco Auto Densi-flow is suitable. For more information see ref. 26.

@ Surface-bound transferrin, for example, is removed by washing the cells twice in 280 mM sucrose, 25 mM citric acid, 24.5 mM sodium citrate, pH 4.6 [96].

© Up to 10 cycles with the ball-bearing homogenizer may be needed (depending on cell type and/or HM composition), up to 15 strokes of the pestle of a Dounce homogenization or up to 20 passages through a syringe needle should be sufficient. Always monitor the progress of homogenization by phase contrast microscopy.

(6 Alternatively allow a discontinuous gradient of equal volumes of 5, 10, 15 and 20% iodixanol to diffuse overnight. See ref. 26 for information on making continuous gradients.

© Satisfactory separation of endosomal compartments from other cells or tissues may require modulation of either the density gradient or the

Table 5.5 Preformed iodixanol gradients for analysis of endocytosis

Cell/tissue Input1 Grad2

g hours Fractionation3

Analysis

Ref.

CHO pns 5-20 90000 20.0 EE, LE, TGN, LDL-derived cholesterol 97

(25RA) PM trafficking

HeLa pns 5-20 125000 20.0 EE, RE, PM, Degradation of EGF 98

lys receptor pns 5-20 90 000 18.0 EE, RE, PM Rab11 endocytic targeting 99

MDCK pns 5-20 90000 18.0 EE, RE, PM, Transferrin endocytosis 96

NRK pns 12.5-30d 113000 1.5 EE Caveolin recruitment to 100

early endosomes

Renal BB4 mic 15-25 100000 3.0 CP, endosomes Myosin localization 101

1 pns = post-nuclear supernatant; mic = microsomal fraction.

2 Gradient density range in % iodixanol; d = discontinuous.

3 EE = early endosomes; LE = late endosomes, RE = recycling endosomes, TGN = trans Golgi network, PM = plasma membrane; CP = coated pit; lys = lysosomes.

4 BB = brush border.

centrifugation conditions. This can only be determined by trial and error.

® Use of other centrifugation conditions (shorter times at higher RCFs for example) should be checked against the recommended parameters for their efficacy.

@ For gradient collection low-density end first, use either the tube puncture device of the Beckman Fraction Recovery System to deliver a dense liquid such as Maxidens™ to the bottom of the tube or the Labconco Auto Densi-flow device to aspirate from the meniscus. For collection dense-end first, use tube puncture. More information about gradient collection can be found in ref. 26.

© In this gradient the density of plasma membrane, lysosomes, recycling endo-somes and early endosomes increases in that order[96]. Some other published papers that have reported the use of similar gradients are given in Table 5.5.

Analysis of clathrin-coated vesicle processing in self-generated iodixanol gradients; endocytosis of asialoglycoprotein by rat liver [102,103]

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