Team:NJU-China/Protocol
Protocol
1、Thermal shock transformation of ZYCY10P3S2T E.coli
(1) Culturing ZYCY10P3S2T E.coli in PSⅠ liquid until OD≥4;
(2) Mix 50μL culture liquid with 100ng plasmid (100ng/μL) and place on ice for 30min;
(3) 42℃ metal bath heat hit 90-120s;
(4) Place the mixture on the ice again for 5min;
(5) Add 400-500μL sterile antibody free medium, shake the shaker for 40min.
2、Extraction of plasmid DNA from E.coli
Take 1L bacterial solution as an example：
(1) Collect E.coli by centrifugation, 8000rpm, 20min;
(2) Add 20ml Solution Ⅰ, 40ml SolutionⅡ and 30ml Solution Ⅲ in turn, and mix them well;
(3) Centrifuge at 8000rpm for 20min at 4℃, filtrate, and then supernatant into a new centrifuge bottle;
(4) Add 0.6 times volume of isopropyl alcohol to precipitate overnight at -20℃;
(5) 8000rpm, 20min, centrifugation at 4℃;
(6) Discard the supernatant, add 10mL P1, 10ml P2 and 10ml P3 solution in turn, and mix well;
(7) Centrifuge at 8000rpm for 20min at 4℃ and filter supernatant into the new tube;
(8) Add 4ml ER to each tube and mix them upside down;
(9) Add 0.3 times the volume of isopropyl alcohol, mixed and precipitated at -20℃ for 1h;
(10) Add 2.5mL BL solution to the adsorption column, centrifuge at 6000rpm for 1min;
(11) Continuously add the mixed liquid in (9) to the adsorption column, centrifuge, and discard the waste liquid;
(12) Add 10mL ED into the adsorption column, centrifuge at 6000rpm for 1min;
(13) Add 10ml PW to the adsorption column, and centrifuged at 6000rpm for 1min;
(14) Add 10ml PW to the adsorption column, and centrifuged at 6000rpm for 1min;
(15) Centrifuge at 7000rpm for 2min, put the adsorption column into a new collection tube, open the cover and dry it;
(16) Add 1mL ddH20 to the adsorption column at 65℃, stood for 5min, and centrifuged at 6000rpm for 2min;
(17) Add ddH20 in the collection tube to the adsorption column again, stood for 1min, then centrifuged at 7000rpm for 2min;
(18) Measure the concentration of plasmid in the collection tube and stored at -4℃.
3、Cell transfection
Liposome mediated transfection
(1) Take a 12-well plate and add the cells to be transfected into the well one day in advance (10%DMEM);
(2) Discard the solution in the pore plate when the cell quantity is 1×10^6 / well;
(3) Solution 1: Mix 60μL Lipofectamine 2000 with 600μL OPti and let stand for 10min;
(4) solution 2: mix 2μg plasmid, 100μl opti, 100μl solution 1 and let stand for 20min;
(5) Add 100μL solution 2 and 900μL opti to each well;
(6) Inducer was added 2h later, and 2%DMEM was changed 6h later.
Electric transfection
(1) Electric parameters: C:950μF, V:250V, PC: 250OPM, Vol:800μL (containing 2×10^6 cells), Cuvette: 4mm, plasmid :10μg in each transfer cup;
(2) Shock twice and distribute evenly into two wells of 12-well plate, and add 600μ L 10%DMEM (Horse Serum);
RNA was collected 24h after culture, and proteins or exosomes were collected 36h after culture.
4、Agarose gel electrophoresis
(1) Configure 30ml 1% agarose solution, heat and melt, add 30μL dye EB;
(2) Add 1μL loading buffer with 1μL plasmid in each well（also 1μL Marker）;
(3) 120V electrophoresis for 30min;
(4) Fluorescence chromogenic photography.
5、Detected cytotoxicity By CCK8 assay
(1) Same as cell transfection (the transfected plasmid/liposome was considered to contain cytotoxicity), cells were considered to have fully absorbed the toxicity after 8h;
(2) Dispense 100μl of cell suspension(6000cells/well) in a 96-well plate;
(3) Incubate the plate for an appropriate length of time(e.g.,12,24,36,48 hours) in the incubator;
(4) Add 10μl of CCK-8 solution to each well of the plate;
(5) Measure the absorbance at 450nm using a microplate reader.
6、RNA Detection by q-RT-PCR
Harvest total RNA from cells or tissues
from cells:
(1) Discard culture media from the culture dish; (2) Wash the cell with 2ml PBS once to make sure culture media is completely removed; (3) Add 1ml TRIzol reagent every 1 x 10^6 cells; (4) Pipette several times to permit complete dissociation of the cell. Transfer the liquid to a clean 1.5ml tube; (5) Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for homogenization. Vortex vigorously to mix thoroughly. Place 2-3min at room temperature; (6) Centrifuge at 16000 x g at 2-8℃ for 15 minutes; (7) Remove the aqueous phase of the sample by angling the tube at 45° and pipetting the solution out to a clean 1.5ml tube. (8) Add 0.5ml of 100% isopropanol to the aqueous phase, per 1ml of TRIzol Reagent; (9) Incubation at -20℃ for at least one hour; (10) Centrifuge at 16000 x g at 2-8℃ for 20 minutes; (11) Remove the supernatant from the tube, leaving only the RNA pellet; (12) Wash the pellet, with 1ml of 75% ethanol per 1ml of TRIzol Reagent; (13) Vortex the sample briefly, then centrifuge the tube at at 16000 x g at 2-8℃ for 10 minutes; (14) Vacuum or air dry the RNA pellet for 5-10 minutes. (15) Resuspend the RNA pellet in RNase-free water; (16) Store at -20℃ for a short time or store at -70℃for long time preservation.
from tissues:
(1)Take a piece of tissue, put it into EP tube, add 400μ L TRIzol and two steel balls, grind 60 seconds, 60HZ on the grinder; (2)Add 600μl TRIzol; (3)The remaining steps are the same as ‘From Cells’.
q-RT-PCR
(1) Reverse Transcription for siRNA by Vazyme
(a) Components: total volume 10μL
10×RT Mix 1μL, Hiscript Enzyme Mix 1μL, Primer（2μM） 1μL, total RNA 1.5μg, RNase free ddH20 up to 10μL;
(b) Cycling Conditions:
Step 1: 25℃, 5min
Step 2: 50℃, 15min
step 3: 85℃, 5min Step 4: 4℃, infinite
(2) Reverse Transcription for mRNA by Takara
Oligo d(T) 18 can be used as primers as mRNA 3’ prime end with a poly(A) tail;
(a) Components: total volume 10μL ;
5× AMV buffer 2μL, AMVase 0.5μL, dNTPs mixture(10mmol) 1μL, Oligo d(T) 18(50μM)(10×) 0.5μL RRI(40U/μl) 0.25μL, RNA 2μg, DEPC up to 10μL;
(b) Cycling Conditions:
Step 1: 16℃, 15min
Step 2: 42℃, 60min
step 3: 85℃, 5min Step 4: 4℃, infinite
(3) qPCR for siRNA by Vazyme
(a) Components: total volume 20μL
2×miRNA Universal SYBR qPCR Master Mix 10μL, Primer F(10μM) 0.4μL, Primer R(10μM) 0.4μL, cDNA 1μL, ddH2O up to 20μL;
(b) Cycling Conditions:
Step 1: 95℃, 5min
Step 2: 95℃, 10s
Step 3: 65℃, 30s (fluorescence detection)
Step2-Step3, 40 cycles (variable, can be up to 45 cycles) Step 4: 95℃, 10s
Step 5: 65℃, 60s
Step 6: 97℃, 1s (Continuous)
(4) qPCR for mRNA (a) Components: total volume 20μL 10×buffer 2μL, dNTPs mixture (10mmol) 0.4μL, MgCl2 1.2μL, Taq 0.3μL, Sense primer (10mM) 0.5μL, Antisense primer (10mM) 0.5μL, dye 1μL, ddH2O 13.1μL, cDNA 1μL; (b) Cycling Conditions: Step 1: 95℃, 5min Step 2: 95℃, 15s Step 3: 60℃, 30s Step 4: 72℃, 30s (fluorescence detection) Step2-Step4, 40 cycles (variable, can be up to 45 cycles)
Data Analysis: The Comparative Ct Method (ΔΔCT Method) CT---cycles when the reaction reach the threshold, the relative expression level of each miRNA compares to endogenous Control can be described as 2-ΔCT, (ΔCT= CT sample- CT endogenous control). U6, a housekeeping gene, is usually used as endogenous Control for miRNA. And for mRNA, just replace U6 with GAPDH. GAPDH, a housekeeping gene, is usually used as endogenous Control for mRNA.
7、protein Detection by Western Blot
Harvest protein from cells or tissues
from cells:
(1) Adherent cell (e.g., LA-4): suck and discard culture medium, and add 80μl RIPA (pyrolysis fluid) into each whole. Suspend the adherent cells in the whole with a cell scraper. Wash once with 1ml absolute ethanol and three times with 1ml PBS Buffer between scrapes. Absorb the fluid into EP tubes after it is fully mixed; (1*) Suspension cell (e.g., El-4): Transfer the culture medium into 1.5ml tubes. Centrifuge for 4 min at 1k rpm and discard the upper clear liquid (add PBS to wash if there are too many cells). Add 80μl RIPA into each tube; (2) Crack it with ultrasound and leave it alone on ice for 30 min; (3) Centrifuge for 20 min at 12000x g at 4°C. Absorb the upper clear liquid to a new tube and add 5x SDS to 1/4 of its volume; (4) Metal bath for 5 min at 99°C to make denature protein; (5) Measure the concentration of the protein with BCA method and preserve it at -80°C.
from tissues:
(1) Cut off tissue pieces, about the size of mung beans, and add 80-100μL of RIPA according to the specific tissue size. (2) The remaining steps are the same as ‘From Cells’.
Western Blot
(1) Prepare Solutions;
(2) Gel Electrophoresis; (a) Load protein and molecular weight marker; (b) Add running buffer; (c) Electrify: set the voltage at 80V before the sample reach the dividing line between stacking gel and resolving gel, switch to 120V until the blue stain reach the buttom line; (3) Blotting I. Transfer (a) Carefully cut the gel; (b) Assemble the transfer cassette; (c) Install the cassette and electrify to transfer the protein to PVDF; II. Chemiluminescene (a) Blocking: 5% skimmed milk incubate for 1h; (b) Incubate with diluted primary antibody; (c) Wash membrane: TBST 15min *4; (d) Incubate with diluted secondary antibody; (e) Wash membrane: TBST 15min *4; (f) Add Chemiluminescene substrate; (g) Exposure.
8、Incubating cells with exosomes
Exosomes (100 μg) loaded with siRNAs were incubated with LA-4 cells (10^6 cells). After 24h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of siRNA and mRNA, and for total protein isolation and subsequent western blotting analysis of protein.
9、Modeling and administration of asthma mice
Our modeling strategy is that as followed: female BALB/c mice will be intraperitoneally injected with 50mg OVA with 2mg aluminum hydroxide in 200ul PBS on day 0,14,28; mcDNA in liposome will be given every two days on days 30-40 by nasal inhalation; finally, 50mg gOVA in 30ul PBS will be given on days 42-44 by nasal inhalation.
10、IgE ELISA
(1) Before use, mix all reagents thoroughly; (2) Add Cytokine standard diluted 100μL/well to the standard well, add sample 100μL/well to the sample well, add Dilution buffer R 100μL/well to the blank control well, cover with sealing plate membrane, incubate at 37℃ for 90min; (3) Discard liquid from the well, add 1×Washing buffer 300μL/well; After staying for 1min, discard the liquid in the hole, and repeat 4 times; (4) Add Biotinylated antibody working solution 100μL/well, cover with sealing plate membrane, and incubate at 37℃ for 60min; (5) Repeat step (3); (6) Add streptavidin-HRP working solution 100μL/well, cover with sealing plate membrane, incubate at 37℃ for 30min; (7) Repeat step (3); (8) Add TMB 100μL/well and incubate at 37℃ for 5-30min under dark conditions until the color of the well turns dark blue; (9) Add Stop solution 100μL/well quickly to Stop the reaction; (10) Read the value at 450nm 10min after termination.