Team:SUSTech Shenzhen/Notebook

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Cell culture

1. Prepare DMEM medium containing 10% FBS

2. Set the cell culture condition as 5% CO2 concentration, 37 ℃.

Cell counting

1. Mix 10 μ L cell suspension and 10 μ L 4% typany blue solution evenly, and add them to counter star cell counting plate.

2. Count cells with cell counter

Cell passage

1. Remove the original culture medium with vacuum pump

2. Wash the cell surface with 1 * PBS solution

3. Digest the Intercellular cell matrix with typsin-EDTA solution so that it does not adhere to the wall.

4. Wash the cells with DMEM medium of twice the volume and collect them into 2ml centrifuge tube.

5. Centrifugate with 200xg speed for 3min, and then remove the supernatant.

6. The cells were resuspended with 1ml medium and passed on in the proportion of 1:10.

Cell medium preparation

Mix the filtered 10% FBS with DMEM medium, and preheat the medium to 37 ℃ before use.

Construction of plasmid vector (molecular clone)

a.polymerase chain reaction(PCR)

Amplification PCR system

1.ddH20 15μL

2.10xTaq Buffer with K2SO4 or (NH4)2SO4 2.5μL

3.dNTPs Mix(2mM) 2.5μL

4.Primer-F 1μL

5.Primer-R 1μL

6.MgCl2(25mM) 1.5μL(sometimes integrated in the 10xTaq Buffer)

7.Taq DNA Polymerase(5ng/μL) 0.3μL

8.DNA template 1μL

in total 25μL per sample

Amplification PCR program

(1).95°C pre-heating for 2min

for 35 cycles:

(2).95°C dunaturation 15s

(3).55°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min (5)72°C for fully elongation, 10min

(6) set 4°C for preservation

b.Restriction endonuclease reaction

1. 1mg plasmid corresponds to 1ul restriction endonuclease

2.10x restriction endonuclease buffer

3.ddH2O to make the reaction system reach 30μL per sample

c.Gibson assembly system(2-3 fragments)

1.Total amount of fragments：0.02-0.05 pmols*X μL

2.Gibson Assembly Master Mix(2X) 10μL

3.Deionized H2O 10-X μL

in total 20μL

4. 37°C for 30min, 4°C for preservation

d. Preparation of nucleic acid gel

1. 1% agarose

3. Heat to complete melting

2. Add 10000x nucleic acid dye (genegreen) after cooling to below 80 ℃.

3. Pour the liquid gel into the mold and select the size of sample hole reasonably

4. cooling to nucleic acid gel solidification

(5. Add 6xDNA loading dye to the sample and mix evenly)

e. Nucleic acid gel extraction by kit

1. Column balance steps: add 500 μ l of balance solution BL to the adsorption column CA2, centrifuge at 12000rpm for 1min, pour out the waste liquid in the collection pipe, and put the adsorption column back into the collection tube.

2. cut the single purpose DNA strip from the agarose gel and let it weigh in a clean centrifuge tube.

3. Add the equal volume solution PN to the gel mass and place it in a 50 ° C water bath until the gel is completely dissolved.

4. Add the solution obtained in the previous step into an adsorption column CA2, place it at room temperature for 2min, centrifuge it at 12000rpm for 60s, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube.

5. Add 600 μ l wash buffer PW to the adsorption columnCA2, centrifuge at 12000rpm for 60s, pour out the waste liquid in the collection tube, and put the adsorption column CA2 into the collection tube.

6. Repeat step 5.

7. Put the adsorption columnCA2 into the recovery header and centrifuge at 12000rpm for 2min.

8. Put the adsorption column CA2 into a clean centrifuge tube, add a proper amount of elution buffer EB to the suspended drop in the middle of the adsorption membrane, and place it at room temperature for 2min. DNA solution was collected by centrifugation at 12000rpm for 2min.

f.Plasmids transformation

1. Thaw DH5 α cells on ice, 100 μ l per tube

2. Add 10 μ l linkage system to the competent cells and incubate on ice for 30 minutes.

3. Put the conversion system into a 72 ° C water bath and heat shock for 70s.

4. Put the heat stimulated transformation system on ice immediately for 5min.

5. Add 1ml LB medium and culture at 37 ° C for 1-2h

6. Centrifuge 12000xg for 2min, and reserve 100 μ l LB medium

7. Fully resuspend and smear the cells onto the LB solid antibiotic plate.

Clone screen

1. The monoclonal colonies were selected from the corresponding resistant culture plates and cultured in a centrifuge tube containing 50μL LB medium for a short time.

2. Construct colony PCR system and run the program

Colony PCR system

1. 2*mix 5μL

2.Primer-F 0.4μL

3.Primer-R 0.4μL

4. LB medium containing target colony 1μL

5.ddH2O 3.2μL

in total 10μL

Colony PCR program

(1).95°C pre-heating for 5min

for 25-30 cycles:

(2).95°C denaturation 15s

(3).55°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min (5)72°C for fully elongation, 10min

(6) set 4°C for preservation

3. based on the UV imaging results of nucleic acid gel, the positive samples were preliminarily determined.

4. Sequence the target plasmid with colony as sample

Fig:single colon on antibiotic agar plate

Cell Transfection by using lipo3000

1. Seed cells to be 70%-80% confluent at transfection.

2. Dilute Lipofectamine 3000 reagent in OPTI-MEM medium, mix well

3. Prepare master mix of DNA by diluting DNA in OPTI-MEM medium, then add P3000 reagent, mix well.

4. Add diluted DNA to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio)

5. Incubation in room temperature for 15min.

6. Add DNA-lipid complex to cells.

7. Visualize/analyze transfected cells after 24h.

Flow cytometry operation

1. Start the flow cytometer and initialize the program.

2. Clean the flow cytometer with ddH2O and NaClO

3. Set corresponding flow detection template

4. For the sample adding operation, the cell suspension needs to be filtered before being added to the flow cytometer.

5. Run "dailyclean" and clean the flow cytometer again with ddH2O and NaClO.

Microplate reader operation

1. Set the format of enzyme plate, sample position and detection area

2. inlet plate

3. oscillation

4. Select the light-emitting mode (absorbance / chemiluminescence)

5. Export and analyze results

Fig:Microplate reader

Luciferase (hGluc)-substrate fluorescence reaction

1. Add 10 μ l of cell supernatant after light exposure

2. Prepare hglluc substrate (60x) and dilute it with luciferase substrate dilution buffer.

3. Add 60 μ l diluted luciferase substrate to each 96 well standard enzyme hole.

CCK8 detection reagents-cells fluorescence reaction

1. Wash, digest and resuspend cells

2. Add 10 μ l CCK8 detection regent to each wall of 96 well plate.

3. Add 100 μ L cell suspension to 96 well plate.

4.Incubation at 37 ° C for 2h

5. Use microplate reader to measure 450nm absorbance and analyze data

Fig:cck-8 detection reagent with cells

Mycoplasma test

1. Take cell culture supernatant as PCR template

2. Set up the experimental group, positive control (Mycoplasma) and negative control (H2O), and use the specific primers related to Mycoplasma to carry out PCR reaction.

Mycoplasma test PCR program

(1).98°C pre-heating for 1min

for 30 cycles：

(2).98°C denaturation 10s

(3).58°C for annealing 30s

(4).72°C for elongation in the speed of 1kb/min, 1min

(5)72°C for fully elongation, 10min

(6) set 4°C for preservation

Enzyme-linked immunosorbent ( ELISA) operation(For IL-10、IL-8)

1.Prepare all reagents and samples to room temperature before use, It os recommended that call samples and standards be assayed in duplicate.

2.Remove unused microplate strips from the plate frame, return them to the foil pouchcontaining the desiccant pack, and reaseal.

3. Plate washing: wash the plate with 1x washing buffer (300 μ L / well) for three times and pat the enzyme plate dry.

4. Sample incubation: add standard sample and sample to be tested, 100 μ L / well, incubation at room temperature for 2h

5. Plate washing: discard the liquid in the well, add 1x washing buffer (300 μ L / well) to wash the plate for three times, and pat the enzyme plate dry.

6.Detection antibody incubation: add the detection antibody pre configured to the working concentration into the enzyme plate, 100 μ L / well, mix well, and incubate at room temperature for 1h.

7. Plate washing: discard the liquid in the hole, add 1x washing buffer (300 μ L / well) to wash the plate for three times, and pat the enzyme plate dry.

8.Add the prepared substrate to the enzyme plate, mix it with 200 μ L / well, and incubate at room temperature in dark for 20min.

9. Termination: Add 20 μ L / well termination solution to the enzyme plate, and gently shake the enzyme plate until the color is uniform.

10.Read 450nm absorbance value within 20min

Fig:Enzyme-linked immunosorbent

RNA extraction(QIAGE RNeasy Mini Kit)

1.Cells:harvest a maxium of 1x10^7 cells, as a cell pellet or direct lysis in the vessel.Add the appropriate volumn of Buffer RLT

2.Add 1 volumn of 70% ethanol to the lysate, and mix well by pipetting.

3.Transferup to 700μL of the sample, including any precipitate, to an RNeasy Mini spin colun placed in a 2mL collection tube. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

4.Add 700μL Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

5.Add 500μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15s at 8000xg. Discard the flow-through.

6. Add 500μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2min at 8000xg. Discard the flow-through.

7.Place the RNeasy spin column in a anew 1.5mL collection tube. Add 30-50μL RNase-free water directly to the spin column membrane. Closed the lid, and centrifuge for 1min at 8000xg to elute the RNA.

Plasmids extraction

1. Pour 1.5 ml overnight grown culture in a microcentrifuge tube. Centrifuge at room temperature (or 4°C) for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min).

2. Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.

3.Add 500 μl ice cold STE solution. Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting. Centrifuge at 4°C for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min). Remove the supernatant from the tube completely.

4.Add 350 μl of STET solution and resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.

5.Add 250μl of freshly prepared solution of lysozyme and mix immediately by vortexing for 5 seconds.

6.place the tube in a boiling water bath for approximately 1 min Centrifuge the tube at maximum speed (14,000 rpm) in a microcentrifuge for 10 min at 4°C or room temperature.

7. Transfer the supernatant containing plasmid promptly in new microcentrifuge tube.

8. Add equal volume of Phenol:Chloroform:Isoamylalcohol (25:24:1) in the supernatant. Mix by vortexing for 10 sec. Centrifuge at maximum speed at 4°C. Transfer the supernatant to fresh microcentrifuge tube.

9. Add equal volume of isopropanol in the supernatant. Mix it by inverting the tube 4 – 6 times. Centrifuge at maximum speed (14,000 rpm) for 30 min at 25°C. Remove the supernatant completely.

10. Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times. Centrifuge at 14,000 rpm (maximum speed) for 10 min at 25°C. Remove the supernatant completely.

11. Dissolve the pellet in 25 μl sterile double distilled water or TE

Reverse transcription PCR program

(1). 37°C for 15min

(2). 85°C for 5s

(3). 4°C for preservation

Real time polymerase chain reaction (qPCR)

qPCR reaction system

2xqPCR mix 10μL

Forward primer (10 µM stock) 0.4μL

Reverse primer (10 µM stock) 0.4μL

PCR grade water 4.2μl

qPCR program

(1).Initial denaturation 94°C 2min

For 40 cycles in total

(2).denaturationn 94°C 15s

(3).Annealing, extension, and read fluorescence 60°C 1min

3D cell culture dishes preparation

1. Prepare 1.2% agarose gel

2. add 550μL hydrogel into each well of 24-well plate.

3. before the gel is solidified, 3D printing mold is placed in the middle of well to make the dent needed for cell precipitation.

4. after the gel was solidified, the mold was pulled out, adding 200μL per hole DMEN diluted penicillin and streptomycin to incubate 2h for subsequent use.

3D cell mass preparation

1. Add 1.5 * 10 ^ 5 cells to each well.

2. After 24 hours of culture, observe the cell morphology, discard the supernatant, wash the floating cells and adherent cells with 1 x PBS solution, and add DMEM medium for culture again.

3. Repeat the above steps until the cells form a good mass.

Cell migration experiment

1. Choose IL-8 as the cytokine to be verified.

2.Set up negative control (bilateral culture medium), experimental group (IL-8 and culture medium are located on both sides respectively), positive control (bilateral IL-8).

3.Keep tracking and photographing for 15 hours to obtain data and express the movement rule of cells in migration experiment.

Fig:transwell assay operation

Fig:group set in cell migration experiment

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