4. Khakh, B.S. et al., Pharmacol. Rev., 53, 107
(2001).
5. Ding, Y. et al., J. Auton. Nerv. Syst., 81, 289
(2000).
6. Le, K.T. et al., J. Neurosci., 18, 7152 (1998).
7. Robertson
stain at final concentrations of 2 x 10-6 M PKH2 dye
and 1 x 107 cells/ml in a 2 ml total volume, perform the
followingusing aseptic techniques:
1. Adherent or bound cells must first be removed
using
cold PBS.
13. Pellet the Cells by Centrifuging at 2000 x g for 5 minutes at 4º C.
14. Aspirate supernatant carefully .
15. Resuspend the cell pellet with cold PBS. (1.0 ml per 1 x 107
cold PBS.
13. Pellet the Cells by Centrifuging at 2000 x g for 5 minutes at 4º C.
14. Aspirate supernatant carefully .
15. Resuspend the cell pellet with cold PBS. (1.0 ml per 1 x 107
Repeat the Wash Step once again without
counting the cells.
7. Add 1-2.5 mL of the prepared 1× Extraction
Buffer A per 2-5 × 107 cells. Incubate on ice for
10-15 minutes.
8. Homogenize
References
1. Vaughn, J.L., Goodwin, R.H., Tompkins, G.J., and McCawley,
P. (1977). The establishment of two cell lines from the insect
Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13,
213-
Small or valuable (1 to 100 :l or 1 mg) Procedure #1
Moderate
(3 to 100 mg tissue, 103-107 mammalian cells or 109 bacterial cells)
Procedure #2
Large (0.1 to 1.0 g tissue or 107-108 mammalian cells
(25-300 °C): 85 x 107 per °C
Strain point: 505 °C
Annealing point: 548 °C
Dielectric Constant: 1 Kc 7.6 D.C.
Volume Resistivity (Ohm-cm): 25 °C 6.5 x 1012
Lost tangent: 1 Kc 2.0% 100 Kc