Haynes Lab:Notebook/Jan/2016/02/13: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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== | ==Thawing Cell Lines on 2/13/16== | ||
* | * Thawed one vial of U2OS cells from box A1 and one vial of U2OS 6-3 cells from box B6 according to the following protocol: | ||
*Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath. | |||
*After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood. | |||
*Transfer 5 mL of 100% FBS into a 15 mL conical tube (one per frozen cell vial). | |||
*Retrieve the frozen cell vial from the -150°C freezer. | |||
*Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed. Do this for no more than 2 minutes. | |||
*Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do not mix by pipetting. | |||
*Spin the cells + FBS at room temperature at 1000 rpm for 3 minutes. If you have just one tube, use a counter balance of equal volume (6 mL water). | |||
*Get a new T-25 flask and label it with the label it with the cell line name, your initials, the date, "thawed" to indicate that this is a new culture. | |||
*After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet. | |||
*Flick the tube to gently resupend the pellet in the ~100 uL FBS. | |||
*Stand the t-25 flask up and remove the cap. | |||
*Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times). | |||
*Transfer the cells + medium into the T-25 flask. | |||
*Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion. | |||
*Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight. | |||
*Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste. | |||
Latest revision as of 01:33, 27 September 2017
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Thawing Cell Lines on 2/13/16
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