Here is a documentation of the protocols we have used in the wet lab for our project.
Preparations
Feeling state bacterial preparation and transformation
1. Inoculate DH5α bacteria into 5ml LB medium to active.
2. Add actived bacteria liquor into LB medium proportionally, ( 1 percent blend of bacteria liquor required in 50ml LB medium), then incubate at 37℃ until OD600=0.5
3. Take 25ml liquor into 50 ml sterile centrifuge tube which is precooled, centrifuge at 4,000rpm for 10 minutes at 4℃
4. Remove the supernatant, add 15ml 0.1mol/L CaCl2 (precooled) to resuspend, put the tube on the ice for 30 minutes.
Then, centrifuge at 4,000rpm for 10 minutes at 4℃
5. Remove the supernatant, add 10ml 0.1mol/L CaCl2 (precooled) to resuspend, put the tube on the ice for 30 minutes.
Then, centrifuge at 4,000rpm for 10 minutes at 4℃
6. Remove the supernatant, add 1ml 0.1mol/L CaCl2 containing 15% glycerin to resuspend
7. Distribute feeling state bacteria liquor 100μl per EP tube
Plasmid transformation
1. Put the Feeling state bacteria on the ice for 20 minutes
2. Add 10μl plasmid liquor , then put the mixture on the ice for 30 minutes
3. Heat at 42℃ for 90s
4. Put on ice immediately for 5 minutes
5. Pour the mixture into 1ml LB culture medium, incubate at 37℃ for 1h
6. Pour 100μl bacteria liquid to do the spread plate, incubate at 37℃ until bacterial colonies grow on the surface of medium.
Cell Culture
Tissue culture
The Flp-InTM T-RExTM 293 Cell Line (Human Embryonic Kidney cells that contain a single stably integrated FRT site at a transcriptionally active genomic locus, and stably expressing the tetracycline repressor protein) was purchased from Thermo Fisher Scientific (R78007). Cells were cultured in a humidity controlled chamber at 37°C with 5% CO2 in media containing DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1 unit/ml penicillin, 1 μg/ml streptomycin, 2 mM L-glutamine and 1X MEM non-essential amino acids.
Transient transfection
293 cells were seeded at a density of 0.05 x 106 cells per well of a 24-well plate and cultured under standard conditions overnight. The following day, the cells were transfected with plasmid constructs using Lipofectamine 2000 (Thermo Fisher) as per manufacturer’s protocol.
Cell passage
1. Discard medium, Add 5ml PBS into the culture bottle, move the dish slightly to wash away rest waste medium, discard the PBS.
2. Add 1ml trypsin solution (0.25%trypsin + 0.02%EDTA) to the culture, digest for 2 minutes at 37℃.
3. Add another 4ml PBS to end the reaction, Blow slightly to flush down cells from bottom wall until cell evenly distributed.
4. Transfer the suspension into 15ml centrifuge tube, centrifuge at 1,000×g for 5 minutes
5. Discard the suspension, resuspend the cell with 2ml medium, blow slightly until cell evenly distributed(blow 50 times enough), next, proportional distribute in culture bottoms(different cell culture in different radio).
6. Add another medium to complement culture system to 5ml.
Exosome
Mycoplasma Detection
First . use PCR Master Mix to amplify gene fragments.
Second . Agarose Gel Electrophoresis (AGE)
I. 1% Agarose Gel Preparation.
A. Dilute 1g agarose in 100mL TAE buffer.
B. Heat until the solution become clear and transparent in the microwave oven.
C. Cool down the solution to 60℃.
D. Then, add EtBr（5mg/L）proportionally (1:10000) into the mixture, shake the conical flask until fully dissolve.
E. Pour the solution into the mixing tank, wait for 30-40min.
II. Start electrophoresis. Operating voltage 100V, 40 minutes
III. Analysis in a gel doc.
Construct exosome-booster stable cell line
I. lentivirus package
(we use GM easy TM Lentiviral Packaging Kit to package lentivirus)
1) one day before the transfection, passage the 293T cell by a ratio of 1:4 into 10cm culture dish, prepare to transfection when cell density up to 80%.
2) medium exchange before transfection:
exchange the cell medium into fresh medium two hours before the transfection (12ml per culture dish)
note: 293T cell is weak in adherence, please notice operate slightly to avoid blowing off cells.
3) transfection
①prepare the transfect reagent according to the upper system at 1.5ml centrifuge tube.
②Keep the mixture still standing at normal temperature for 20 minutes, next, drop the system into the cell culture
uniformly, then, incubate at 37℃.
4) medium exchange:
6 hours later, change the medium into 15ml medium without antibiotics, then, incubate at 37℃.
Note: there would be a small amount of lentivirus generated, please notice that the medium and all the pipette tips should be treated before discard.
5) lentivirus collection:
48h later, collect the culture medium, centrifuge at 500×g for 5 minutes at 4℃.Then, filtrate the supernatant by 0.45μm filter.
6) distribution and storage:
distribute the lentivirus liquid 50μl per centrifuge tube, store at -80℃.
II. Transfect 250μl virus liquid into 293T cell culture medium
III. Exchange mixed medium into complete medium after 8 hours’ incubation
IV. Adding 2μl/ml puromycin into the culture when there are a great quantity of cells on the surface
1. Collect culture solution, distribute in the centrifuge tube, centrifuge at 10,000×g for 30 minutes to obtain the supernatant
2. Transfer the supernatant into the ultracentrifugation tube, centrifugate at 13,000×g for 90 minutes at 4℃
3. Discard the supernatant, resuspended by PBS solution, centrifugate at 13,000×g for 2h at 4℃
4. Discard the supernatant, resuspended by 30μL PBS solution
Transmission electron microscope anlysis
1. The method of hydrophilizing copper carrier:
be exposed to ultraviolet rays for one night
2. Add 10μL sample on the copper carrier, precipitate for one minute, use filter paper to drain off the extra liquid from the edge of copper carrier.
3. Drop 10μL 2% phosphotungstic acid on the copper carrier, set aside for one minute, use filter paper to drain off the extra liquid from the the edge of copper carrier.
4. Dry at room temperature for 2 minutes
5. Observed under transmission electron microscope(operating voltage is 200KV)
WB
Reagent preparation
1. TBST buffer: 20ml 1.0M Tris-HCl (pH 8.0), 8.8g NaCl , 0.5ml Tween 20, 800ml ddH2O
2. Transfer buffer: 2.9g Glycine, 5.8g Tris, 0.37g SDS, 200ml methanol, dilute with ddH2O to 1000ml
3. Blocking buffer: 5g skim milk, 100ml TBST, store at 4℃
4. Luminol western blot ECL plus is purchased from Milipore company
protein extraction
PBS(precooled at 4℃, 0.01M pH7.2～7.3), 1.5mlEP tube×2, High speed refrigerated centrifuge precooled at 4℃
I. Take the cell culture out of the incubator. Discard the culture medium
II. Add 5ml PBS into the culture dish(100mm), move the dish slightly to wash away the rest waste medium , discard the PBS. Repeat this step for once.
III. Add 1ml lysis buffer into cell culture dish. Wait for 2 minutes. Blow slightly to flush down cells from bottom wall until cell evenly distributed.
IV. Remove the mixture into EP tube, blend vertically at 4℃ for 20 minutes. Centrifuge at 16,000rpm for 5 minutes at 4℃
V. Distribute the supernatant into 0.5ml centrifuge tubes. Store at －80℃.
SDS-PAGE
30% Acrylamide, 10% SDS, 10% APS, TEMED, 1×SDS-PAGE buffer, 1.5mol/L Tris-HCl(pH8.8), 1.0mol/L Tris-HCl (pH6.8), 5XSDS-PAGE Loading Buffer
I. Protein Quantification and preparation: Measure the protein extractive concentration by nanodrop. Take 100μg protein sample, add loading buffer by a ratio of 5:1. Heat at 100℃ for 5 minutes.
II. Assemble the gel freeze equipment.
III. 5 ml 12% separating gel preparation (1.6ml H2O, 2.0ml 30% Acrylamide, 1.3ml 1.5mol/L Tris-HCl (pH8.8), 0.05ml 10%SDS, 0.05ml 10%APS, 0.002ml TEMED)
IV. After add 0.002ml TEMED, blend the liquor. Then, add the separating gel into the glass gap immediately, add another 1ml ddH2O to block. Set aside at normal temperature for 40 minutes.
V. 2ml 5% stacking gel preparation.(1.4ml H2O, 0.33ml 30% Acrylamide, 0.25ml 1.5mol/L Tris-HCl(pH8.8), 0.02ml 10%SDS, 0.02ml 10%APS, 0.002ml TEMED)
VI. Pour the upper ddH2O and add the stacking gel. Carefully insert comb into gel sandwich until bottom of teeth reaches top of front plate. (Tilting the comb at a slight angle is helpful for insertion without trapping air bubbles). Still standing at normal temperature for 40 minutes.
VII. After finish freeze. Use ddH2O wash the comb hole.Then assemble the SDS-PAGE equipment
VIII. Add 100μg heat treated protein sample into the hole, start electrophoresis.(operate voltage 150V), 40 minutes.(until the front of bromophenol blue reaches the bottom of gel) Take down gel and do continued Western Blot analysis.
(Note: 5μL protein sample with Coomassie brilliant blue R250 is required in another gel to verify the existence of protein.)
Western Blot analysis
I. Submerge the gel in the ddH2O for 5 minutes, repeat the step for three times. Then, transfer the gel into the transfer buffer, wait for 10 minutes.
II. PVDF membrane should be soaked in formaldehyde for 5 minutes,then, submerge all the materials including membrane, filter paper and sponge pad in the transfer buffer for 10 minutes. The size of membrane and filter paper should be close.
III. Put sponge pad on the negative pole, add two filter papers, gel, PVDF membrane , two filter paper and another sponge pad in order, squeeze out bubbles.
IV. Put the equipment in the electrophoresis chamber, add electrophoresis buffer, operate current 200mA, 100 minutes. (note: electrophoresis chamber should be put in the ice to cool down )
V. When electrophoresis finish, move PVDF membrane into blocking solution and stay overnight at 4℃.
VI. Primary antibody incubation: Discard blocking solution, add 5ml diluted primary antibody(1:5000), incubate one hour at normal temperature . Next, recover antibody, wash with 5ml TBST for four times, each time last 10 minutes.
VII. Secondary antibody incubation: Discard TBST buffer, immediately add diluted secondary antibody (1:10000), incubate one hour at normal temperature. Recover antibody, wash with 5ml TBST for four times, each time last 10 minutes.
VIII. Imaging: Prepare ECL plus(1:1 mix reagent A and reagent B in the dark chamber, put the mixture on the ice). CCD camera precool at -25℃，drop ECL plus on the gel, then, imaging and reserve the results.
Confocal microscopy analysis
1. Exosome staining:
Exosomes were labeled with PKH26, according to the manufacturer’s protocol, with some modifications. Briefly, exosome pellets were resuspended in 1 mL Diluent C. Separately, 1 mL Diluent C was mixed with 4 μL PKH26. The exosome suspension was mixed with the stain solution and incubated for four minutes. The labeling reaction was stopped by adding an equal volume of 1% BSA. Labeled exosomes were ultracentrifuged at 100,000 ×g for 70 minutes, washed with PBS, and ultracentrifuged again.
2. Prepare cancer cell slides(cells should be incubated with cytoskeletal protein β-tubulin beforehand for 6h)
3. Incubate cancer cells with stained-exosome medium for 6 hours
4. Do laser scanning confocal microscopy
Scratch Assay
Transfect or other intervention cells, 6-well plates, after 36h-48 hours of transfection, use a 200μl pipette tip to keep the pipette vertical and consistent. Rinse PBS 3-5, and observe the scratches under the microscope, with or without floating cells. Change to serum-free medium/1% FBS medium. At 24h and 48h, the floating cells were washed away with PBS, replaced with fresh serum-free medium/1% FBS medium, and photographed by inverted microscope. Image J analyzes cell crawling and finally uses Graphpad 6.0 to calculate cell crawling.
Transwell
The Transwell test steps are as follows:
A. The 8μm pore size chamber was selected for experiment. In the Invasion group, 60 μl of Matrigel gel (1:8) was needed, and the chamber was incubated for 30 min at 37 °C. The chamber of the Migration group was not coated.
B. Stark transfection or other intervention cells in serum-free medium for 12h, trypsinize the cells, neutralize, centrifuge, resuspend in serum-free/1% FBS medium, count, and adjust the cell concentration (1*105-5 *105/ml), blow evenly, carefully join the chamber, do not shake the chamber vigorously.
C. Add 600μl of 10%-20% FBS culture solution to the lower chamber, and put the chamber into the hole with tweezers. Observe the air bubbles under the chamber. Lift the chamber with bubbles, remove the bubbles at the edge of the hole, and put them into the hole. Operate gently to avoid violent shaking of the orifice or chamber.
D. After 24h-36h, 4% paraformaldehyde was fixed for 20min, washed twice with PBS, the residual formaldehyde was washed away, dyed with 0.3% crystal violet dye solution for 30min, crystal violet was recovered, the chamber was cleaned, and the cotton was removed with tweezers. Do not touch the cells under the membrane and then dry.
E. Photograph analysis, the chamber was placed on an empty glass slide, inverted white light microscope observation, and images were taken. Count with Image J software, and finally calculate the cell proliferation using Graphpad 6.0.
Parts Construction
Primer design
Software: Snape
Notes:
1. Forward primer 5’ end add XbaI restriction enzyme cutting sites
Reverse primer 3’ end add SpelI restriction enzyme cutting sites
2. Tm=60℃
3. All the parts fragments must test restriction enzyme cutting sites, make sure there is no EcoRI, PstI, XbaI and SpelI sites.
Plasmid Extraction
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
2. Centrifuge at 10,000 x g for 1 minute at room temperature.
3. Decant or aspirate and discard the culture media.
4. Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
(Note: RNase A must be added to Solution I before use.)
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
6. Add 250 µL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
(Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.)
7. Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.
(Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation.)
8. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
9. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
Optional Protocol for Column Equilibration:
a. Add 100 µL 3M NaOH to the HiBind® DNA Mini Column.
b. Centrifuge at maximum speed for 30-60 seconds.
c. Discard the filtrate and reuse the collection tube.
10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
11. Centrifuge at maximum speed for 1 minute.
12. Discard the filtrate and reuse the collection tube.
13. Add 500 µL HBC Buffer.
(Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 6 for instructions.)
14. Centrifuge at maximum speed for 1 minute.
15. Discard the filtrate and reuse collection tube.
16. Add 700 µL DNA Wash Buffer.
(Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use.)
17. Centrifuge at maximum speed for 1 minute.
18. Discard the filtrate and reuse the collection tube.
(Optional: Repeat Steps 16-18 for a second DNA Wash Buffer wash step.)
19. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
(Note: It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.)
20. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
21. Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
(Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5.)
22. Let sit at room temperature for 1 minute.
23. Centrifuge at maximum speed for 1 minute.
(Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.)
24. Store DNA at -20°C.
PCR
Use PCR Master Mix to amplify gene fragments. Each part do three parallel repetitions
Agarose Gel Electrophoresis (AGE)
1. 1% Agarose Gel Preparation.
A. Dilute 1g agarose in 100mL TAE buffer.
B. Heat until the solution become clear and transparent in the microwave oven.
C. Cool down the solution to 60℃.
D. Then, add EtBr（5mg/L）proportionally (1:10000) into the mixture, shake the conical flask until fully dissolve.
E. Pour the solution into the mixing tank, wait for 30-40min.
2. Start electrophoresis. Operating voltage 100V, 40 minutes.
3. Analysis in a gel doc. Finally, cut off the target band and put in a 2ml centrifuge tube, store at 4℃.
DNA Extraction
(use Omega Gel extraction Kit to extract DNA )
1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running bufferas its pH will increase and reduce yields.
2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4. Add 1 volume Binding Buffer (XP2).
5. Incubate at 50-60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6. Insert a HiBind® DNA Mini Column in a 2 mL Collection Tube.
7. Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind® DNA Mini Column.
8. Centrifuge at 10,000 x g for 1 minute at room temperature.
9. Discard the filtrate and reuse collection tube.
10. Repeat Steps 7-9 until all of the sample has been transferred to the column.
11. Add 300 µL Binding Buffer (XP2).
12. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
13. Discard the filtrate and reuse collection tube.
14. Add 700 μL SPW Wash Buffer.
(Note: SPW Wash Buffer must be diluted with 100% ethanol prior to use.)
15. Centrifuge at maximum speed for 1 minute at room temperature.
16. Discard the filtrate and reuse collection tube
17. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
(Note: It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications.)
18. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
19. Add 15-30 μL Elution Buffer or deionized water directly to the center of the column membrane.
(Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on pH. If eluting DNA with deionized water, make sure that the pH is around 8.5.)
20. Let sit at room temperature for 2 minutes.
21. Centrifuge at maximum speed for 1 minute.
(Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.)
22. Store DNA at -20°C.
Enzyme Digestion
1. Use Nanodrop to measure the concentration of gene fragments and plasmids
2. Calculate digestion system’s concentration
3. Put the system in the Thermostatic Water Bath at 37℃ for 4h-12h
4. Add 10×lodding Buffer to end the reaction
5. Agarose Gel Electrophoresis , operating voltage 100V, 40 minutes
6. Imaging in gel doc. Cut off the target band. DNA extraction.
(Note: As mRFP fragment is located between Spel site and Xbal site, two bands would be obtained, cut off linear plasmid fragments band in which represents the length is around 2000bp.)
Enzyme Connection
1. Connect the linear plasmid fragment and target gene fragment using T4 ligase offered by Takala company, following is the system component:
(Note: the proportion of linear plasmid fragment and target gene fragment should between 1 to 3 and 1 to 10.)
2. Keep at 4℃ for the whole night or keep the system at 16℃ for 4 hours
Plasmid Transformation and Function Verification
1. Put the feeling state bacteria on the ice for 2-5 minutes to melt.
2. Add 10μl part plasmid liquor , then put the mixture on the ice for 30 minutes
3. Heat at 42℃ for 60-90s
4. Put on ice immediately for 5 minutes
5. Blend the mixture with 1ml LB medium, incubate at 37℃ for 1h
6. Pour 100μl bacteria liquid to do the spread plate , after 30 minutes’ standing , incubate at 37℃ until bacterial colonies grow on the surface of medium.
7. Pick white clonal strain into 5ml LB culture medium with 30μg/μL chloramphenicol, (Set four parallel repetitions.) and shake overnight at 37℃.
8. Extract plasmid. Sequencing the products fragment with VF and VR primer to confirm the specificity and reliability.
