Trypsinize or scrape? - (Jul/13/2007 )

1.
I would like to harvest adherent cells(MCF7)and have nucleotides contain measured by HPLC. Would you recommend sraping (possible damage to cells) or trypsin (poisoned cells detach much easily, I am not sure if trypsinisation cannot influence ATP levels in different way)?

2.
At which g would you recommend to harvest? I dont wont to damage the cells but I would like to get all of them (and normalize ATP on protein content).

Tx for replies in advace
karpaton

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Q1: a third possibility is to lyse directly from the plate/flask; as trypsination is more stress, you may alter nucleotide level f.i. by exhausting ATP; so scraping off is better than trypsination

Q2: x1.670 rpm f.i. in a blue cap

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Mechanical separation (Scraping, trituration / repetitive aspiration via aspirating pipet, etc.) is more likely to rupture the cell membrane, but enzymatic debridement (trypsin, papain, etc.) chops up surface proteins, of which some are signalling molecules of one form or another, of which I am certain that some cytoplasmic portions of complexes are transcription factors in addition to their other functions (beta-catenin, for instance)... The bottom line being, you're "stuck" either way... (Ha ha)

If you're looking at cyclic AMP or ATP levels, or really anything, for that matter, you want to minimize as much "harvest- induced changes" as possible. Proteolytic/ enzymatic release of the cells - I'm guessing by trypsin or papain, the two "standard" cysteine proteases - will surely cause alterations in the surface protein expression and/ or cytoplasmic signalling in your MCF7's. Without sounding like a professor (which I am not) or sounding like a pompous buffoon (no comment on my likeness), I'll suggest washing the cells with as benign a solution as possible (DPBS is dulbecco's phosphate buffered saline, so it's only good if you're not looking at phosphates and/ or kinase activity -- I'd suggest HBSS or something with the osmolarity you seek but little else) and, unless I misunderstood your application, harvest the cells straight in the flask/ culture dish. Why risk the chance of adding noise when you can lyse and harvest the contents of the cells right then and there? Just my two cents, so take it with a grain of salt, but if your end goal is to harvest the cells, then lyse them anyhow - why add the step of surface dissociation that could alter the ATP levels you are looking for? Apologies if that came out poorly - I just wanted to make it clear and wasn't sure I made sense.



Again, I may be totally misunderstanding your desired application, so take this with a grain of salt, but if you harvest the cells right in the flask after several washes, (harvesting via Tween-20, Triton X-100, or simply mechanical disruption, whatever) you won't have to worry about getting every last cell, worry of cell damage and untimely lysis, or of excessive protein background. If I missed the boat on the application, I apologize. As such, I'll answer anyhow -- MCF7's are not as fragile as some lines, but they're no Vero, either... without rambling, the standard "g's" for intact cell harvests, at least as I've seen them in literature, are all ~200xg for 5 to 10 minutes, depending on volume, at 5*C (+- 3*C). I'm guessing that your volume would be from ~10 mL to ~250 mL per vessel... same speed but increased time if there's more volume per vessel. The decreased temperature slows metabolism in the cell, by the way, in case you were wondering why not room temperature.

I hope this helps you more than it confuses, as I'm new to this forum and have a tendency to ramble something fierce...
Kind regards,
pjgramp

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MCF7:s... You don´t need to neither trypsinize nor scrape them. They will come off if you just pipette up and down a few times in for instance PBS or your isolation buffer.

D

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