Team:NJU-China/Experiments/Protocol
Protocol
Plasmid transfection and extraction of exosomes
HEK 293 cells were cultured in 25 cm2 flasks(Biofil) at 37℃, 5% CO2.When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Tpep and Bcl2 siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. The cell culture medium was then harvested 48 h after transfection, and the exosomes loaded with Bcl2 siRNA were harvested from the medium using an exosome isolation kit (Invitrogen) according to the manufacturer’s instructions. The resulting pellet was then resuspended in PBS.
Western blot
1) Gel Electrophoresis preparation: TEMED,30% Acr-Bis(29:1), 1M tris-HCl(Ph 6.8), 1M tris-HCl(Ph 8.8), APS, 10%SDS
2)Load protein and molecular marker and add running buffer.
3)Electricity: 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 bottom line;(At the same time, precool the transfering buffer at -20℃)
4)Transfer
i carefully cut off stacking gel
ii Assemble the transfer cassette in the order of cassette black side, sponge, filter paper, gel, PVDF, filter paper, sponge, cassette white side.
iii Install the cassette and set the electrify current at 300mA for 90mins to transfer the protein onto PVDF.
5) Blocking: 5% defatted milk incubation for 60mins.
6) Primary antibody incubation overnight at 4℃。
7) Wash membrane: 1 X TBST10mins*5.
8) Secondary antibody incubation for 60mins at room temperature( on the shaker)
9) Wash membrane: 1 X TBST10mins*5.
10) Add Chemiluminescene substrate, incubate in dark for 5min, then expose
2)Load protein and molecular marker and add running buffer.
3)Electricity: 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 bottom line;(At the same time, precool the transfering buffer at -20℃)
4)Transfer
i carefully cut off stacking gel
ii Assemble the transfer cassette in the order of cassette black side, sponge, filter paper, gel, PVDF, filter paper, sponge, cassette white side.
iii Install the cassette and set the electrify current at 300mA for 90mins to transfer the protein onto PVDF.
5) Blocking: 5% defatted milk incubation for 60mins.
6) Primary antibody incubation overnight at 4℃。
7) Wash membrane: 1 X TBST10mins*5.
8) Secondary antibody incubation for 60mins at room temperature( on the shaker)
9) Wash membrane: 1 X TBST10mins*5.
10) Add Chemiluminescene substrate, incubate in dark for 5min, then expose
Mice tissue total RNA extraction by trizol
1. Cut up the tissue to the weight of 50-100mg, add 1ml trizol and and 2 steel balls into 1.5ml RNAase free EP tube. Install the EP tubes in the grinding machine and set the machine running at 60 Hz for 90s, repeat 5 times with a 60s interval.
2. Transfer the grinding fluid into a new EP tube, centrifuge at 10000g for 10mins, discard the sediment, transfer the supernate into a new EP tube.
3. Add 1/5 volume of chloroform, shake vigorously and let it stand at room temperature for 10mins.
4. Centrifuge at 16000g for 10min( precool the centrifugal machine to 4℃)
5. Transfer the supernate( about 500-600ul) to a new EP tube, add isopropyl alcohol in equal volume, let it sit for more than 60mins at -20℃。
6. Centrifuge at 16000g for 10mins, discard the supernate, wash the sediment with 75% DEPC ethyl alcohol.( repeat blowing and beating the sediment)
7. Centrifuge at 16000g for 10mins,discard the supernate, upside down the EP tubes until the ethyl alcohol is volatized.
8. Add DPEC water to solve the sediment( The volume of DEPC water depends on the sediment, usually 20ul-50ul)
9. Measure RNA concentration and adjust it to about 1000ng/ul, stored at -80℃。
2. Transfer the grinding fluid into a new EP tube, centrifuge at 10000g for 10mins, discard the sediment, transfer the supernate into a new EP tube.
3. Add 1/5 volume of chloroform, shake vigorously and let it stand at room temperature for 10mins.
4. Centrifuge at 16000g for 10min( precool the centrifugal machine to 4℃)
5. Transfer the supernate( about 500-600ul) to a new EP tube, add isopropyl alcohol in equal volume, let it sit for more than 60mins at -20℃。
6. Centrifuge at 16000g for 10mins, discard the supernate, wash the sediment with 75% DEPC ethyl alcohol.( repeat blowing and beating the sediment)
7. Centrifuge at 16000g for 10mins,discard the supernate, upside down the EP tubes until the ethyl alcohol is volatized.
8. Add DPEC water to solve the sediment( The volume of DEPC water depends on the sediment, usually 20ul-50ul)
9. Measure RNA concentration and adjust it to about 1000ng/ul, stored at -80℃。
RNA reverse transcription and Qpcr
1) RNA reverse transcription
i)Taqman reaction system(10ul system, adjust RNA concentration to 1000ng/ul in advance):
AMV: 0.5ul
AMV buffer:2ul
dNTP:1ul
DEPC water: 4.5ul
Taqman: 1ul
Template RNA: 1ul
Target RNA and reference RNA reaction system need to be constructed individually.
ii) Reverse transcription
Edit the protocol as follows:
16℃ 15mins
42℃ 60mins
85℃ 5mins
4℃ store
2) Qpcr Taqman reaction system(20ul per hole)
10X buffer: 2ul
dNTPs: 0.4ul
MgCl2: 1.2ul
rTaq:0.3ul
ddH2O:14.77ul
Taqman: 0.33ul
cDNA: 1ul
i)Taqman reaction system(10ul system, adjust RNA concentration to 1000ng/ul in advance):
AMV: 0.5ul
AMV buffer:2ul
dNTP:1ul
DEPC water: 4.5ul
Taqman: 1ul
Template RNA: 1ul
Target RNA and reference RNA reaction system need to be constructed individually.
ii) Reverse transcription
Edit the protocol as follows:
16℃ 15mins
42℃ 60mins
85℃ 5mins
4℃ store
2) Qpcr Taqman reaction system(20ul per hole)
10X buffer: 2ul
dNTPs: 0.4ul
MgCl2: 1.2ul
rTaq:0.3ul
ddH2O:14.77ul
Taqman: 0.33ul
cDNA: 1ul
Mammalian cell total protein isolation by RIPA lysis
Reagents:
RIPA lysis (medium) (Beyotime, P0013C);
PMSF (100mM) (Beyotime, ST506);
SDS-PAGE Loading Buffer (6 x) (Beyotime，P0015F);
0.25% Trypsin (1X), Phenol Red(Life Technologies，15050-065);
Protocol:
(1) Discard the culture media;
(2) Wash the cell with 2ml PBS once to make sure culture media is completely removed;
(3) Add 1-2 ml 0.25% Trypsin, incubate at 37℃ for 2min，add 5-6 ml culture media to stop the digestion when cells are dissociating quickly;
(4) Pipette several times to permit complete dissociation of the cell. Transfer the cells to a clean 15ml tube;
(5) Centrifuge at 800rpm at room temperature for 3min;
(6) Add 1 μL PMSF to RIPA lysis every 100 μL;
(7) Discard the supernatant, add 1ml RIPA lysis every 75cm2 culture dish surface area;
(8) Incubation on ice for 30 min;
(9) Centrifuge at 10000-14000 x g for 3-5min;
(10) Transfer the supernatant, add 1 μL SDS-PAGE loading buffer every 5μL supernatant;
(11) Boil to denature the protein at 99℃ for 5min, store at -20℃;
RIPA lysis (medium) (Beyotime, P0013C);
PMSF (100mM) (Beyotime, ST506);
SDS-PAGE Loading Buffer (6 x) (Beyotime，P0015F);
0.25% Trypsin (1X), Phenol Red(Life Technologies，15050-065);
Protocol:
(1) Discard the culture media;
(2) Wash the cell with 2ml PBS once to make sure culture media is completely removed;
(3) Add 1-2 ml 0.25% Trypsin, incubate at 37℃ for 2min，add 5-6 ml culture media to stop the digestion when cells are dissociating quickly;
(4) Pipette several times to permit complete dissociation of the cell. Transfer the cells to a clean 15ml tube;
(5) Centrifuge at 800rpm at room temperature for 3min;
(6) Add 1 μL PMSF to RIPA lysis every 100 μL;
(7) Discard the supernatant, add 1ml RIPA lysis every 75cm2 culture dish surface area;
(8) Incubation on ice for 30 min;
(9) Centrifuge at 10000-14000 x g for 3-5min;
(10) Transfer the supernatant, add 1 μL SDS-PAGE loading buffer every 5μL supernatant;
(11) Boil to denature the protein at 99℃ for 5min, store at -20℃;
Transmission electron microscopy
For transmission electron microscopy analysis, the exosome samples were prepared as described above. Briefly, the exosome pellet was placed in a droplet of 2.5% glutaraldehyde in PBS buffer and fixed overnight at 4 °C. The exosome samples were rinsed 3 times in PBS for 10 min each and then fixed in 1% osmium tetroxide for 60 min at room temperature. Then, the samples were embedded in 10% gelatine, fixed in glutaraldehyde at 4 °C and cut into small blocks (less than 1 mm3). The samples were dehydrated in increasing concentrations of alcohol. Then, the samples were placed in propylene oxide and infiltrated with increasing concentrations of Quetol-812 epoxy resin mixed with propylene oxide for 3 h per step. Finally, the samples were embedded in pure, fresh Quetol-812 epoxy resin and polymerised at 35 °C for 12 h, 45 °C for 12 h, and 60 °C for 24 h. Ultrathin sections were cut using a Leica UC6 ultra-microtome and stained with uranyl acetate for 10 min and lead citrate for 5 min at room temperature. The samples were then observed with a transmission electron microscope (JEM-1010) at a voltage of 80 kV.
Validation in vivo.
To construct a obesity mouse model, we select one month old mice in normal weight, provide them with high-fat diet. The mice were divided into 4 groups, group I was injected with PBS, group II egfr exosome, group III and group IV Bcl2 siRNA- tpep exosome. Exosomes with tpep on the membrane and Bcl2 siRNA inside was injected through caudal vein every two days. The dosage received is 100ul at the concentration of 1000ng/ul. Weight data was collected everyday. Three weeks later, the mice was killed by ether anesthesia, blood was sampled by removing the eyeball, heart, liver, kidney, lung, spleen and adipose tissue was separated for further reaserch.
ITT
Starvation treatment in group IV for 6 hours, intraperitoneally inject insulin 0.75 unit per kg, Took blood sample form tail end and measure blood glucose at the time of 0, 15, 30, 45, 60, 90, 120 mins.
GTT
Starvation treatment overnight and intraperitoneally inject glucose, the following step is the same as ITT
ITT
Starvation treatment in group IV for 6 hours, intraperitoneally inject insulin 0.75 unit per kg, Took blood sample form tail end and measure blood glucose at the time of 0, 15, 30, 45, 60, 90, 120 mins.
GTT
Starvation treatment overnight and intraperitoneally inject glucose, the following step is the same as ITT
Targeting verification in vivo
4 mice were divided into two groups, group I was injected through caudal vein with 100ul egfr siRNA exosome(egfr siRNA 1000ng/ul), group II was injected with 100ul egfr siRNA- tpep exosome. 12 hours later, the mice were dissected and their liver, heart, kidney, spleen, lung and adipose tissue were separated. Total RNA was extracted by trizol and reverse transcription was carried out, then Qpcr was used to compare the egfr expression in adipose tissue and in other tissues.
Nanoparticle tracking analysis (NTA)
Sample preparation: Dilute the sample until the liquid is clear, colorless and transparent, control the particle concentration at 10^7-10^8particles/ml. Usually, when there is less than 100 particles in the capture field, the results will be more convincing.
We use NS300(Malvern) to run the sample, choose appropriate Top-plate and O-ring, use 1ml injection to load 600ul-800ul sample, click start camera, adjust the focus and choose appropriate screen gain and camera level until there is DARK signal on the top right corner of the window. Set up other advanced parameters( usually, keep the original parameter), click start and run.
When the three times measurement is finished, adjust the threshold value left side panel until there are less than 5 blue label in the vision, click OK and export data.
We use NS300(Malvern) to run the sample, choose appropriate Top-plate and O-ring, use 1ml injection to load 600ul-800ul sample, click start camera, adjust the focus and choose appropriate screen gain and camera level until there is DARK signal on the top right corner of the window. Set up other advanced parameters( usually, keep the original parameter), click start and run.
When the three times measurement is finished, adjust the threshold value left side panel until there are less than 5 blue label in the vision, click OK and export data.
Serum characterization
TG test
TG test kit(NanJing JianCheng Bioengineering Institute) method:CPO-PAP
i. Sample preparation: Dilute the serum until it is within the range of linearity.
ii. Operation box(96-well plate)
TG test kit(NanJing JianCheng Bioengineering Institute) method:CPO-PAP
i. Sample preparation: Dilute the serum until it is within the range of linearity.
ii. Operation box(96-well plate)
| blank | standards | sample | |
|---|---|---|---|
| ddH2O(ul) | 2.5 | ||
| Standards(ul) | 2.5 | ||
| Sample(ul) | 2.5 | ||
| working solution(ul) | 250 | 250 | 250 |
| Mix up the solution and incubate it at 37℃ for 5mins, then use microplate reader to measure OD510nm | |||
iii. Formula:
    TG(mmol/L)={((sample OD)-(blank OD))/((standards OD)-(blank OD))}*c
    c is the TG concentration of the standard which is known
HDL-C test
HDL-C test kit
i. Sample preparation: Dilute the serum until it is within the range of linearity.
ii. Operation box(96-well plate)
    TG(mmol/L)={((sample OD)-(blank OD))/((standards OD)-(blank OD))}*c
    c is the TG concentration of the standard which is known
HDL-C test
HDL-C test kit
i. Sample preparation: Dilute the serum until it is within the range of linearity.
ii. Operation box(96-well plate)
| blank | standards | sample | |
|---|---|---|---|
| ddH2O(ul) | 2.5 | ||
| Standards(ul) | 2.5 | ||
| Sample(ul) | 2.5 | ||
| R1(ul) | 180 | 180 | 180 |
| Mix up the solution and incubate it at 37℃ for 5mins, then use microplate reader to measure OD546nm(A1) | |||
| R2(ul) | 60 | 60 | 60 |
| Mix up the solution and incubate it at 37℃ for 5mins, then use microplate reader to measure OD546nm(A2) | |||
iii. Formula:
    HDL-C(mmol/L)={{(S(A2)-S(A1))-(B(A2)-B(A1))}/{(T(A2)-T(A1))-(B(A2)-B(A1))}}*c
        S:sample     B: blank     T: standards     c is the TG concentration of the standard which is known
LDL-C test
    LDL-C test kit, the operation steps are the same as HDL-C test
    HDL-C(mmol/L)={{(S(A2)-S(A1))-(B(A2)-B(A1))}/{(T(A2)-T(A1))-(B(A2)-B(A1))}}*c
        S:sample     B: blank     T: standards     c is the TG concentration of the standard which is known
LDL-C test
    LDL-C test kit, the operation steps are the same as HDL-C test
