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Old Jan 21st 2019, 02:56 PM   #1
ChemicalRat
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Post buffer prep for engineers

Hi guys
I need to prepare an unroofing protocol. However, I haven't made buffers since undergrad (I'm an engineering RA since 2014) and humbly ask for a reference or advice where to start... So far I have the pure powders for glucose, NaCl KCl, MgCl2 and CaCl2 ,and made an order of these,

G5882-10X1ML GLUTARALDEHYDE GRADE I
S5011-100G SODIUM PHOSPHATE MONOBASIC
E6758-100G ETHYLENEDIAMINETETRAACETIC ACID*CELL CUL
P4832-50ML POLY-L-LYSINE SOLUTION BIOREAGENT, 0.01%
H4034-25G HEPES, BIOPERFORMANCE CERTIFIED&
03777-10G ETHYLENE GLYCOL-O,O'-BIS(2-AMINOETHYL)-N
43815-1G DL-Dithiothreitol
A8456-100MG 4-(2-AMINOETHYL)BENZENESULFONYL FLUORIDE
158127-5G PARAFORMALDEHYDE, POWDER, 95%

I was intending on using the following adapted protocol

1. Cells on cover slips were washed once with 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (HEPES)-based Ringer's solution consisting of 155 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM NaH2PO4, 10 mM glucose in 5mM HEPES buffer (pH 7.4), and then with Ca++-free Ringer's solution.
2. Subsequently, the cover slips were soaked for about 10 s in poly-lysine solution (0.5 mg ml−1poly-lysine dissolved in Ca++-free Ringer's solution), and then washed three times for a few seconds each in hypotonic Ringer's solution prepared by mixing one part of Ringer's solution with two parts of distilled water (DW). This induced cell swelling, which enabled the cells to burst easily following ultrasonic stimulation. Immediately after immersing in hypotonic solution, the cells were exposed to a small bubble jet by weak ultrasonic vibration in isotonic KHMgE buffer (30 mM HEPES, pH 7.4, 70 mM KCl, 3 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 0.1 mM AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride))
3. Cells unroofed by the bubble jet were washed briefly in fresh KHMgE buffer and immediately fixed for 10 min with 0.5% glutaraldehyde and 1% paraformaldehyde in KHMgE buffer.
4. Fixed samples were washed twice with KHMgE buffer
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buffer, cells, fixation, ringer's, unroofing



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