Jul 16, 2020
- Melissa Teusel1,
- Irina Titkova1,
- Ina Schmitt1,
- Lena Postawa1,
- Marcel Schilling1,
- Ursula Klingmüller1
- 1Division Systems Biology of Signal Transduction, German Cancer Research Center (DKFZ), INF 280, Heidelberg, Germany
External link: https://doi.org/10.1371/journal.ppat.1008461
Protocol Citation: Melissa Teusel, Irina Titkova, Ina Schmitt, Lena Postawa, Marcel Schilling, Ursula Klingmüller 2020. Huh7.5_SOP. protocols.io https://dx.doi.org/10.17504/protocols.io.biapkadn
Manuscript citation:
Robichon K, Maiwald T, Schilling M, Schneider A, Willemsen J, Salopiata F, Teusel M, Kreutz C, Ehlting C, Huang J, Chakraborty S, Huang X, Damm G, Seehofer D, Lang PA, Bode JG, Binder M, Bartenschlager R, Timmer J, Klingmüller U (2020) Identification of Interleukin1β as an Amplifier of Interferon alpha-induced Antiviral Responses. PLoS Pathog 16(10): e1008461. doi: 10.1371/journal.ppat.1008461
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 07, 2020
Last Modified: July 16, 2020
Protocol Integer ID: 38959
Keywords: Huh7.5 cell lines, cell culture, medium, thawing, freezing,
Abstract
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).
Materials
MATERIALS
Fetal Bovine SerumGibco - Thermo FischerCatalog #10270106
Neubauer Improved (NI) Hemocytometer Life TechnologiesCatalog #22-600- 100
0.05% Trypsin-EDTA, phenol redInvitrogen - Thermo FisherCatalog #25300054
Glutamax (100x)Gibco - Thermo FischerCatalog #35050-061
DMEM, high glucose, no glutamine, no phenol redThermo FisherCatalog #31053028
Bovine Serum AlbuminSigma AldrichCatalog #A9418
Penicillin-streptomycin (P/S)Gibco - Thermo FisherCatalog #15140122
Dimetylsulfoxide – DMSO Merck Millipore SigmaCatalog ##41639-100ML
DPBS w/o: Ca and MgPAN BiotechCatalog #P04-36500
15 ml centrifuge tubes greiner bio-oneCatalog #188271
50 ml centrifuge tubes greiner bio-oneCatalog #227261
Tissue Culture Dish D:150 mmTechno Plastic Products (tpp)Catalog #93150
Tissue Culture Test Plates 6-wellTechno Plastic Products (tpp)Catalog #92006
CRYO.S 2 ML PP ROUND BOTTOM INTERNALTHREAD greiner bio-oneCatalog #122263-2DG
Cool Cell LX BiozymCatalog #210004
Mr. Frosty Thermo Fisher ScientificCatalog #5100-0001
Trypan Blue solution 0.4% for microscopyMerck Millipore SigmaCatalog #935395
Cells
Cells
Huh7.5 cells were kindly provided by Charles M. Rice (The Rockefeller University, NY, RRID:CVCL_7927)
Cells are cultivated in incubator at 37°C and 5 % CO2 incubation and 95 % relative humidity
Medium
Medium
Growth Medium
DMEM
1% Glutamax
10% FCS
1% P/S
Growth factor-depleted Medium
DMEM
1% Glutamax
1% P/S
1 mg/ml BSA
Freezing Medium
70% Growth-Medium
20% FCS
10% DMSO
Thawing of cells
Thawing of cells
Pre-warm growth medium in 37 °C water bath
Thaw the cells in a 37 °C water bath (not completely, there should be a small visible ice clump inside the tube)
Transfer the cells into a 15 ml centrifuge tube containing 9 ml pre-warmed growth medium
To this aim, use 1000 µl pipette and add some medium to cryotube, and mix. With this step the little ice clump should disappear
Transfer all liquid from the cryotube to the centrifuge tube, rinse cryotube again and transfer to centrifuge tube
1000 rpm, Room temperature, 00:03:00 , 130 x g
Aspirate supernatant
Resuspend cells in 10 ml fresh growth medium (~7 times)
Transfer the cells to a 150-mm dish (~2x106) in 25 ml growth medium
Let cells adhere to the surface of the dish over night in incubator
Replace the medium with 25 ml fresh growth medium the next day
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)
Cultivation of cells
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)
Cells are split every 3 to 4 days (e.g. Monday to Friday and Friday to Monday)
On one ~80-90% confluent 150-mm plate, 7-10x106 cells can be expected
Aspirate the growth medium
Wash the cells with 10 ml DPBS once
Detach the cells from surface by adding 0.05% Trypsin-EDTA (3 ml for 150-mm dish)
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
Tap the dish carefully and stop the enzyme reaction of Trypsin-EDTA by adding growth medium containing FCS (7 ml for 150-mm dish)
Resuspend the cells and transfer to centrifuge tube
1000 rpm, Room temperature, 00:03:00 , 130 x g
Aspirate supernatant
Resuspend in 10 ml fresh growth medium (~7 times) to remove all clumps
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
Plate the cells as required for your planned experiment or for maintaining the cell culture
Keeping cells in culture:
4 days: 2x106 cells in 25 ml growth medium / 150-mm dish
3 days: 3x106 cells in 25 ml growth medium / 150-mm dish
For experiments: usually 0.6x106 cells / well of a 6-well plate in 1.5 ml of medium is used
After 24 h, wash cells 3 times with DPBS , add 1.5 ml of growth-factor depleted medium and incubate for 3 h
Stimulate cells and perform experiment
Freezing of cells
Freezing of cells
Count the cells as described above go to step #19
Transfer cell suspension to centrifuge tube
1000 rpm, Room temperature, 00:03:00 , 130 x g
Label cryotubes (name of cell line, number of cells, passage, date and your initials)
Resuspend cells to a density of 2x106 cells / ml in freezing medium
Calculate needed amount of freezing medium (E.g. if freezing 6x106 cells, 3 ml are needed)
Prepare freezing medium, mix well
Resuspend pellet in freezing medium using a serological pipette and transfer to cryotubes (1 ml/tube)
Transfer cryotube to Mr. Frosty or Cool Cell LX, only use room temperature Mr. Frosty or Cool Cell LX, never cold ones
Transfer cells in cryotubes in Mr. Frosty or Cool Cell LX to -80°C
Transfer tubes to liquid nitrogen tank on the next day
Record name of cell line, number of cells, passage, date, your name and location of the tube in liquid nitrogen tank