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Cell culture

  ● Surface Coating (Adsorption method) [go]
  ● Cell Recovery [go]
  ● Transfection [go]

->Download all protocols for cell culture: Here
 
 
Adhesive

  ● Enzymatic curing Protocol [go]
  ● Chemical Curing Protocol [go]
  ● Measuring Adhesion strength [go]

->Download all protocols for Adhesive: Here
 
 
● Surface Coating (Adsorption method)
1. Preparation.
The MAPTrix® concentration should be adjusted for each cell line of interest. Routinely, dilute the solution type MAPTrix® product to 0.1mg/mL concentration with distilled water.

2. Product Dilution.
Filter the distilled water through a 0.2 μm pore filter just prior to use.

3. Coating.
Add 125 μL/cm2 MAPTrix® solution to each well and shake your plate by moving it back and forth, together with an upward and downward motion ~3-4 times to evenly coat the plate surfaces.
Note: The volume to coat should be adjusted for the diameter of the culture plate used.

4. Incubation.
Incubate the plate for 2 hours at 37oC.

5. Removing Solution.
Remove the coating solution by pipetting or Pasteur pipette suction.

6. Washing.
Wash the coated plate with the same volume distilled water and then remove the solution by pipetting or Pasteur pipette suction. Wash the plate one more time with serum-free media in the same way.
Note: Avoid scraping bottom surface.
 
Example)
Plate Culture Area (cm2/well) MAPTrix® Volume (μL/well)
6-Well Plate
12- Well Plate
24-Well Plate
96-Well Plate 		9.6
3.5
1.9
0.75
 1200
440
240
10

Dish Culture Area (cm2/well) MAPTrix® Volume (mL/well)
35mm
60 mm
100 mm		 8.8
21.5
56.7
1.10
2.69
7.09

Flask Culture Area (cm2/well) MAPTrix® Volume (μL/well)
25
80
175		 25
80
175
 3.13
10.00
21.88

Note: The culture area calculated is based on the NUNC brand of products.
 
 
● Cell Recovery
1. All cells for experiments can be collected by trypsinization

2. To harvest growing cells, remove the conditioned growth medium by pippeting or pasteur pippet suction. And then wash the petri diameter 100 mm dish with PBS twice.

3. To detach cell from petri dish, treat cell with 25% trypsin solution for (1~3 min)

4. After confirming cells is detached from the surface of petri dish, harvest cells with growth medium containing 10% FBS to nullify trypsin activity

5. After calculating cell concentration, cells are spread on coated petri dishes, or T-flasks, well plates for your specific cell assay
 
 
● Transfection
1. In a six-well or 35 mm tissue culture plate, seed ~2 x 105 cells per well in 2 ml of DMEM growth medium containing 10% FBS and nonessential amino acids

2. Incubate the cells at 37 °C in a CO2 incubator until the cells are 70-80% confluent. This will usually take 18-24 h

3. Prepare MAPTrix® and DNA solution as followed below. Prepare the each following solution in 12 x 75 mm (diameter, height) sterile tube, respectively:
  - Solution A: prepare 2 μg of DNA (plasmid) in 375 μl of serum-free DMEM (containing nonessential amino acids)
  - Solution B: prepare 12 μl of MAPTrix® solution in 375 μl serum-free DMEM

The minimum ratio for complete binding of MAPTrix® solution with targeted DNA as 4:1 (weight ratio) is recommended

4. To form DNA-Protein complex, combine the two solutions, mix gently, and incubate at room temperature for 15-45 min. The solution may appear cloudy, however this will not impede the transfection

5. Wash the cells once with 2 ml serum-free DMEM

6. For a transfection, add 750 μl serum-free DMEM to tube containing the MAPTrix®-DNA complexes. Do not add antibacterial agents to media during transfection. Mix gently and then overlay the diluted complex solution onto the washed cells

7. Incubate the cells for 5 h at 37°C in a CO2 incubator

8. Add 1.5 ml DMEM with 20% FBS without removing the transfection mixture. If toxicity is a problem, remove the transfection mixture and replace with normal growth medium. Replace it with fresh medium 18-24 h after following start of transfection

9. Do analyze a gene activity from cell extracts 24-72 h after the start of transfection, depending on cell type and promoter activity
 
 
● Enzymatic curing Protocol
1. Prepare the modification buffer comprising 50 mg/ml tyrosinase (Sigma) in 0.1M sodium phosphate buffer, 150 mM NaCl, pH 7.0 containing both 25 mM ascorbic acid and 20 mM sodium borate

2. Dissolve MAPTrix® powder at 10 mg/mL in the modification buffer solution

3. Add Mushroom tyrosinase to above MAPTrix® solution at 50 ㎍/mL of modification buffer solution

4. Shaking the reactant mixture for 1 hour at 25 °C with a simultaneously aeration. The reaction time can increase the content of modification of tyrosin

5. Add 10% acetic acid solution to terminate the enzymatic reaction and stir the mixture for 10 min at room temperature. The volume of 10% acetic acid solution should be the same to the reaction volume

6. After modification, the sample is concentrated by ultrafiltration (MWCO 10 kDa) and dialyzed in 5% acetic acid at 4 oC

7. The amount of DOPA modification may be assessed by matrix-assisted laser desorption ionization mass spectrometry with time-of-flight (MALDI-TOF MS) analysis
 
 
● Chemical Curing Protocol
1. 4 arm PEG-SG is dehydrated and in N2 gas or Ar gas, stored frozen and dissolved immediately before use.

2. MAPTrix® is dissolved at 10 mg in 100 μl of DW or 0.1 N NaOH solution until was homogenous but opaque.

3. 4 arm PEG-SG was quickly dissolved at 20 mg in 100 μl of 100 mM sodium bicarbonate buffer, pH 8.5 -9.0.

4. With vortex mixing MAPTrix® solution, 4 arm PEG-SG solution is dropping into the MAPTrix® solution and mixed evenly.

5. As two solution are mixed, 10 μl of mixture solution is placed on the 1st material to be adhered. The volume can be depended on appropriated adhesive strength and on applied materials.

6. The 2nd material to be adhered is placed on the 2nd material.

7. Fix the material until moderate adhesion is showed.

Note: This protocol is not suitable adhesion in water or in buffer.
 
 
● Measuring Adhesion strength
1. The modified MAPTrix® is dissolved with oxidant solution on the surface of the 1st material you want to adhere.

2. Place the 2nd material to be adhered on the 1st modificed MAPTrix®

3. Place the adhesive surfaces in dry condition or in relative high temperature for at least 24 hrs

4. Once evaporated, it is time to measure the physical properties. Further investigation on the temporal course of MAPTrix® hardening and/or curing is recommended for your test

5. Measure the physical properties when evaporated
 
[Precaution]
1. Once MAPTrix® was treated with tyrosinase, make sure the modified MAPTrix® is freeze-dried and store it in a condition as described below.

2. After once using it, the remaining MAPTrix® should be stored under oxygen-free environments in the room temperature or sealed in -70 oC. For details, refer to the note below.

3. It is hard to handle modified MAPTrix® due to its adhesive character. To avoid loss of modified MAPTrix®, apply directly it to test surface or solution as your convenience.

4. To prevent the oxidation of DOPA, do keep modified MAPTrix® in deep freezer or vacuum environment in the -20 °C. These conditions do not make further oxidation for at least 7 days