Kinebuchi, M., Matsuura, A., Ohya, K., Abo, W. and Kitazawa, J.
Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)