Team:NJU-China/Notebook/Protocol

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1. Transfection of chassis and isolation of exosomes

HEK293 cells were seeded in 225-cm2 flasks (Corning). When the cells reached approximately 70-80% confluence, they were co-transfected with plasmids encoding Lamp2b-iRGD and KRAS 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 MOR 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.
2. 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.
3. Incubating cells with exosomes

Exosomes (100 μg) loaded with KRAS siRNAs were incubated with A549 cells (106 cells). After 24 h incubation, the recipient cells were collected for total RNA extraction and subsequent quantitative RT-PCR analysis of KRAS siRNA and KRAS mRNA, and for total protein isolation and subsequent western blotting analysis of KRAS protein.
4. Mammalian Adherent Cell Total RNA isolation using TRIzol reagent

Reagents:
TRIzol(Life Technologies，15596-026);
DEPC-treated water(Life Technologies，4387937);
75% DEPC ethanol;
PBS;
Chloroform;
Isopropanol;

Protocol:
(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 106 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.
5. microRNA Detection by q-RT-PCR

Reagents:
AMV Reverse Transcriptase XL for RT-PCR (TaKaRa，2630A)
dNTP mixture (TaKaRa，4030);
Taq (TaKaRa，DR100A);
miRNA probe;
GAPDH probe;

Protocol:
(1) Reverse Transcription
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 AMV 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: 42℃, 60min
Step 2: 85℃, 5min
Step 3: 4℃, infinite
(2) qPCR
(a) Components: total volume 20μL
10×buffer 2μL dNTPs mixture (10mmol) 0.4μL MgCl ₂ 1.2μL Taq 0.2μL Sense primer (10mM) 0.5μL Antisense primer (10mM) 0.5μL ddH _O 13.1μL cDNA 1μL
(b) Cycling Conditions:
Step 1: 95℃, 5min
Step 2: 95℃, 30s
Step 3: 95℃, 30s
Step 4: 95℃, 30s (fluorescence detection)
Step2-Step4, 40 cycles (variable, can be up to 45 cycles)
(c) 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). GAPDH, a housekeeping gene, is usually used as endogenous Control for mRNA.
6. 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℃;
7. Western Blot

Protocol:
(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 130V 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% whole 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, incubate in dark for 5min
(g) Exposure
8. Transfection of Mammalian Adherent Cells

Reagents:
Lipofectamine 2000 Transfection Reagent(Life Technologies，11668019);
DMEM;
Opti MEM;
RPMI 1640;
Fetal Bovine Serum;

Protocol:
(1) When the mammalian cells reached approximately 70%-80% confluence, they were transfected with the labelled siRNA;
(2) Transfection components;

(3) Sample, preparation：
(a) Dilute DNA/RNA in serum-free Opti-MEM or reduced serum culture (1:4), mix by pipette gently;
(b) Dilute Lipofectamine 2000 in serum-free Opti-MEM or reduced serum culture (1:4), incubate at room temperature for 5 min;
(c) Mix the diluted DNA/RNA and the diluted Lipofectamine 2000, incubate for 5min;
(4) Discard the culture media；
(5) Wash the cell with 2ml PBS once to make sure culture media is completely removed；
(6) Change the culture media to serum-free Opti-MEM or reduced serum (1%-2%)DMEM/ RPMI 1640；
(7)Add DNA/RNA and Lipofectamine 2000 mixture to culture media, mix thoroughly by gently rotating the culture vessel，incubate at 37°C in 5% CO₂.
(8) Optional: Change to complete culture media 4-6h after transfection.
9. Endotoxin test for exosomes

Reagents:
ToxinSensor Single Test Kit (Genscript)
LAL reagent water
Endotoxin Standard

Protocol:
1. Dilute the endotoxin with LAL Reagent Water of a concentration of 1EU/ml.
2. Prepare a serial of two-fold dilutions from the 1EU/ml endotoxin solution as shown in the following chart.

3. Carefully transfer 0.2ml of positive control (0.015EU/ml), negative control (LAL Reagent Water), and exosomes to the LAL vials. Cap the vials and mix them thoroughly.
4. Incubate all vials at 37℃ in a water bath for 60 minutes.
5. Check whether there is a gel formed or not.
10. in vivo Bioluminescence Imaging

(1) Inject 10μl/g body weight of D-luciferin firefly (15mg/ml in PBS; Caliper Life Sciences Catalog XR-1001 or a similar product from another vendor) into the animal, as described below.
(2) Wait 5 minutes, and then anesthetize the mice by placing it into the gas anesthesia chamber.
(3) Image the mice.