User:Igor R. Kuznetsov/Notebook/RBL-2H3/2010/02/18

RBL-2H3

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Test of adhesion at room temperature Two surfaces: clean glass and BSA-coated glass (exposed to 0.5 g% solution of BSA in Hepes NaCl 300mOsm ph 7.4 for 30 minutes). Two types of media: original growth media (see below), and Hepes as above (centrifuge for 5 min at 1000 rpm, resuspend in Hepes). Growth media: MEM Eagles (cat# Cibco 11095-080) + 10% Huclone Fetal Clone III + 5 mls L-glutamine (200mM)(Gibco Cat. # 25030-081) + 5mls Pen/Strep (5000 units Pen and 5000 mcg Strep) (Gibco Cat # 15140-122) 60 minutes The image below contains 4 columns, with typical cell images after 60 minutes of adhesion at room temperature. First column shows cells in growth media on clean glass, second - cells in growth media on BSA glass, third - cells in hepes on clean glass, and fourth - cells in hepes on BSA glass. KS: - buffer cell looks fine to me – you can see the filopodia! - first two for buffer + BSA look fine too. The third one looks like a lysed cell, which is not good. - I have never seen RBLs on hepes. Hence I can only guess. The filopodia look a bit funny to me but as long as the membrane is intact they might be OK. - hepes+BSA: The top cell might have some membrane issues and the bottom cell looks funny as well. 180 minutes The image below contains 4 columns, with typical cell images after 180 minutes of adhesion at room temperature. First column shows cells in growth media on clean glass, second - cells in growth media on BSA glass, third - cells in hepes on clean glass, and fourth - cells in hepes on BSA glass. KS: - buffer: First one lysed second one might be OK - buffer+BSA: Both are lysed hence dead. - hepes: don’t look happy either - hepes + BSA: top one looks really funcky – never seen this before, and bottom one might be OK.