Human embryonic kidney HEK293T cells are cultured in 10 cm dishes with DMEM plus 10% CS in 5% CO2 atmosphere at 37°C. Mouse embryonic fibroblasts are incubated in DMEM containing 10% FCS, 1% sodium pyruvate solution and 1% non-essential amino acids. Cells are splitted rationally when reaching around 80%
confluence. For splitting, cells are washed once with 1xPBS and trypsinized by 1 ml trypsin/EDTA for 2 min at 37°C. Serum containing medium is added to stop trypsin digestion and cells are centrifuged for 3 min at 800 rpm. Afterwards medium supernatant is aspired and cells are re-suspended and seeded in new dishes.
5.1.2 Cell counting
For certain experiments, proper number of cells is required. Therefore, cells are counted prior to seeding. After trypsin digestion, centrifugation and re-suspension described in culture section, 25μl cell suspension is added into 5 ml filtered CasyTon, mixed well by pipetting up and down. Subsequently, cells are counted by Casy Model TT according to the manufacturer’s manual.
Alternatively, cell density can be measured by the Neubauer commercial counting chamber. Put the glass cover on the Neubauer chamber central area, introduce 10μl cell
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suspension to the edge of glass cover, then place the Neubauer chamber under the microscope and start counting in the four squares. In the end, cell density is calculated via the following formulas, cell density=number of cells*10000/4.
5.1.3 Transfection
Before transfection, the clean mini- or midi-preparation of plasmid should be prepared and the concentration is measured by Nanodrop spectrophotometer.
For HEK293T cells, the day before transfection, cells are splitted 1:5or at other rational ratio. To prepare transfection mixture, add 500μl sterile ddH2O into a 15 ml tube, then add a certain amount of plasmids(1-5 μg) and 500μl 2x HBS. During vortexing, 50μl 2.5 M CaCl2 is added drop wise, followed by an incubation time of 15 min. During that time, 10μl 25 mM chloroquin, which inhibits the acidification of the lysosomes and therefore counteracts the degradation of the inserted DNA, was added to the cells.
Subsequently, the transfection solution was drop wise added to the cells. Cells were put back to the incubator. 6 - 8 h later, medium was changed with fresh cell culture medium and cells were further cultured for 24-48h until use.
For MEFs, transfection is performed either using lipofectamin 2000 or polyplus, according to the manufacturer’s manual. siRNA was transfected with lipofectamin 2000 according to the manufacturer’s manual. To determine the transfection efficiency, 24-48h after transfection, cells were used for further analysis.
5.1.4 Thawing and freezing
Cells were thawed quickly at 37°C in the water bath. Afterwards, added 4ml fresh medium to mix the cell suspension, and transferred to a 15 ml tube, centrifuged for 3
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min at 800 rpm. Subsequently, suck out the supernatant and re-suspended cells in 10 ml fresh medium and transferred to a prepared culture dish. For refreezing, semi-confluent or confluent cell cultures were chosen. After washing, trypsinizing, and centrifugation, cells were re-suspended in 1 ml refreezing medium and transferred to cryogenic vials.
Vials were cooled slowly overnight in the isopropanol box at -80°C. Next day, vials were transferred to liquid nitrogen for permanent storage.
5.1.5 Treatment of cells prior to adhesion assay
Cells were starved overnight in DMEM containing 0.5% CS before adhesion assays.
After trypsination, soybean trypsin inhibitor was used to stop trypsin digestion. Cells were transferred to a 15 ml tube and centrifuged for 3 min at 800 rpm. The supernatant was removed and cells were washed one with PBS. Subsequently, cells were re-suspended in suspension medium (DMEM + 0.25% BSA) and incubated in suspension for 45min at 37°C, before they were seeded for the further experiments.
5.1.6 Adhesion assay
A 96-well plate was coated with 3 μg/ml human fibronectin overnight at 4°C. Next day, the wells were blocked with DMEM supplemented with 0.25 % BSA for 1 hour at 37 °C.
The starved cells were kept in suspension for 45min and seeded in the wells (for NIH3T3 cells, 2.5x 104 cells/well) for the indicated time periods. Non-adherent cells were washed away by PBS and the remaining cells were fixed for 15 min in 4% PFA.
After three times washing, cells were stained with0.1 % crystal violet in 0.1 M borate buffer for 1h under shaking. Excessive crystal violet was removed by three times washing with 1x PBS, OD550 was measured by the spectrophotometer.
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5.1.7 Isolation of Genomic DNA from Eukaryotic Cells
The isolation of genomic DNA from mouse embryonic fibroblasts was conducted using PureLink Genomic DNA Kit, according to manufacturer’s instructions.
5.1.8 Lentivirus Production and Generation of Stable Cell Lines
The lentiviral vectors pLKO.1, pMD2.G and psPAX2 were purchased from Addgene.
HEK293T cells were transfected with 7 μg pMD2.G, 10 μg psPAX2 and 13 μg pLKO.1 harboring the corresponding shRNA, respectively, by standard calcium-phosphate co-precipitation. Seventy-two hours after transfection, the virus-containing supernatant was collected, cleared by centrifugation for 7 min at 2000 rpm, and sterile-filtrated.
HEK293T cells were directly incubated with the corresponding lentiviral supernatant for 24 hours and afterwards selected with fresh cell culture medium containing 0.4 μg/ml puromycin. Control cells were generated via transduction with virus encoding for the empty pLKO.1.
5.1.9 Whole cell lysates
To obtain whole cell lysates, cells were lysed by addition of proper amount of RIPA buffer and scraped from the cell culture dish using a rubber police man, then collected into 1.5ml tubes. Chromosomal DNA and cell debris were pelleted by addition of 50 μl sepharose beads (5 min rotation on the rotator) and centrifugation (13000 rpm, 20 min).
The supernatant was mixed with 2x or 4x SDS sample buffer and boiled for 5 min at 95°C. To prepare sepharose beads: Sephadex G10 beads were suspended in Triton buffer at the volume ratio1:2.
5.1.10 Quantification of surface integrin expression by flow cytometry.
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Integrin α5 (clone MFR5) and integrin αV (clone RMV7) antibodies are purchased from BD Biosciences. Integrin β1 (clone HMβ1-1) and integrin β3 (clone 2C9.G2) antibodies are obtained from Biolegend. Secondary antibodies (biotin-SP-conjugated goat α-rat IgG), streptavidin–FITC and rhodamine Red-X-AffiniPure Goat Anti-Armenian Hamster IgG (H+L) are purchased from Jackson ImmunoResearch. For quantification of surface integrin expression, cells are trypsinized and mixed with fresh cell medium, after centrifugation, the supernatant is carefully removed and cells are re-suspended in suspension medium (DMEM containing 0.25% BSA) and incubated for 45 min at 37°C.
Then, 2 × 105 cells are incubated with appropriate primary antibodies (diluted 1:300) in FACS buffer (5% heat-inactivated FCS, 1% sodium azide in PBS) for 1 h at 4°C.
After three times washing with FACS buffer, secondary antibodies are applied for 1h at 4°C. If it is necessary, cells are washed three times again and the fluorescent antibody is applied for 1h at 4°C. Afterwards, cells are washed again and re-suspended in FACS buffer. Samples are analyzed by flow cytometry (LSRII, BD Biosciences), the mean fluorescence intensity is analyzed.
5.1.11 Generation of target gene knockout cells by CRISPR-cas9 method
3x104 Cerulean positive NIH3T3 cells are seeded in 0.1% gelatin coated 24-well plate.
Next day, transfection is performed using plasmids harboring Cas9 and gRNAs targeting cerulean and gene of interest. After transfection, when cells are confluent, cells are transferred to gelatin-coated 6-well plate or even 10cm dishes and cultured for another 7-10 days. Afterwards, cells are sorted through flow cytometry and cerulean negative cells are collected in 96-well plate at the density of 1 cell/well, then cultured
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at 37 °C, 5% CO2. When cells are enough for subsequent analysis, western blot test or FACS measurement is carried out to verify the knockout cells.