TITLE: Huh7.5_SOP
AUTHORS: Melissa Teusel, Irina Titkova, Ina Schmitt, Lena Postawa, Marcel Schilling, Ursula Klingmüller

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">SOP for thawing, cultivation and freezing of the Huh7.5 cell line by the Division Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ).</div></div></div>

[STEPS]
?. [Medium]
Growth MediumDMEM1% Glutamax10% FCS 1% P/S
?. [Thawing of cells]
Pre-warm growth medium in  water bath
37 °C
?. [Thawing of cells]
Thaw the cells in a water bath (not completely, there should be a small visible ice clump inside the tube)
37 °C
?. [Thawing of cells]
Transfer the cells into a 15 ml centrifuge tube containing 9 ml pre-warmed growth medium
?. [Thawing of cells]
Transfer all liquid from the cryotube to the centrifuge tube, rinse cryotube again and transfer to centrifuge tube
?. [Thawing of cells]
Centrifuge: 1000 33, 3 min
?. [Thawing of cells]
Aspirate supernatant
?. [Thawing of cells]
Resuspend cells in 10 ml fresh growth medium (~7 times)
?. [Thawing of cells]
Transfer the cells to a 150-mm dish (~2x106) in 25 ml growth medium
?. [Thawing of cells]
Let cells adhere to the surface of the dish over night in incubator
?. [Thawing of cells]
Replace the medium with 25 ml fresh growth medium the next day
?. [Thawing of cells]
Split after 2-3 days as described in "Cultivation of cells" (check for confluency to be around 80% to 90%; for example: thawn on Thursday, change medium on Friday and split on Monday)
?. [Thawing of cells]
To this aim, use 1000 µl pipette and add some medium to cryotube, and mix. With this step the little ice clump should disappear
?. [Medium]
Growth factor-depleted MediumDMEM 1% Glutamax1% P/S1 mg/ml BSA
?. [Medium]
Freezing Medium70% Growth-Medium20% FCS10% DMSO
?. [Cultivation of cells]
Cells are used for experiments until passage 25 (total passage number starting from the stocks provided by Charles M. Rice, The Rockefeller University)
?. [Cultivation of cells]
Cells are split every 3 to 4 days (e.g. Monday to Friday and Friday to Monday)
?. [Cultivation of cells]
On one ~80-90% confluent 150-mm plate, 7-10x106 cells can be expected
?. [Cultivation of cells]
Aspirate the growth medium
?. [Cultivation of cells]
Wash the cells with 10 ml DPBS once
?. [Cultivation of cells]
Detach the cells from surface by adding 0.05% Trypsin-EDTA (3 ml for 150-mm dish)
?. [Cultivation of cells]
Place the cells with Trypsin-EDTA in an incubator and incubate for 3 to 4 minutes by which the cells should be detached from the surface
?. [Cultivation of cells]
Tap the dish carefully and stop the enzyme reaction of Trypsin-EDTA by adding growth medium containing FCS (7 ml for 150-mm dish)
?. [Cultivation of cells]
Resuspend the cells and transfer to centrifuge tube
?. [Cultivation of cells]
Centrifuge: 1000 33, 3 min
?. [Cultivation of cells]
Aspirate supernatant
?. [Cultivation of cells]
Resuspend in 10 ml fresh growth medium (~7 times) to remove all clumps
?. [Cultivation of cells]
Dilute 50 µl of cells 1:2 using medium, count the cells applying a 1:2 dilution with Trypan blue using a Neubauer improved hemocytometer
?. [Cultivation of cells]
Plate the cells as required for your planned experiment or for maintaining the cell culture
?. [Cultivation of cells]
Keeping cells in culture:4 days: 2x106 cells in 25 ml growth medium / 150-mm dish3 days: 3x106 cells in 25 ml growth medium / 150-mm dish
?. [Cultivation of cells]
For experiments: usually 0.6x106 cells / well of a 6-well plate in 1.5 ml of medium is used
?. [Freezing of cells]
Count the cells as described above
?. [Freezing of cells]
Transfer cell suspension to centrifuge tube
?. [Freezing of cells]
Centrifuge: 1000 33, 3 min
?. [Freezing of cells]
Label cryotubes (name of cell line, number of cells, passage, date and your initials)
?. [Freezing of cells]
Resuspend cells to a density of 2x106 cells / ml in freezing medium
?. [Freezing of cells]
Transfer cryotube to Mr. Frosty or Cool Cell LX, only use room temperature Mr. Frosty or Cool Cell LX, never cold ones
?. [Freezing of cells]
Transfer cells in cryotubes in Mr. Frosty or Cool Cell LX to -80°C
?. [Freezing of cells]
Transfer tubes to liquid nitrogen tank on the next day
?. [Freezing of cells]
Record name of cell line, number of cells, passage, date, your name and location of the tube in liquid nitrogen tank
?. [Freezing of cells]
Calculate needed amount of freezing medium (E.g. if freezing 6x106 cells, 3 ml are needed)
?. [Freezing of cells]
Prepare freezing medium, mix well
?. [Freezing of cells]
Resuspend pellet in freezing medium using a serological pipette and transfer to cryotubes (1 ml/tube)
?. [Cells]
Huh7.5 cells were kindly provided by Charles M. Rice (The Rockefeller University, NY, RRID:CVCL_7927)
?. [Cells]
Cells are cultivated in incubator at 37°C and 5 % CO2 incubation and 95 % relative humidity
?. [Cultivation of cells]
After 24 h, wash cells 3 times with DPBS , add 1.5 ml of growth-factor depleted medium and incubate for 3 h
?. [Cultivation of cells]
Stimulate cells and perform experiment