JRD244TRANSWELL experiment

Transwell Experiment Transwell Experiment :

A class of transparent cup-shaped devices; Its shape is a small cup that can be placed in a hole plate, a permeable film having a cup underlayer (typically a polycarbonate film). The membrane microporous size is between 0.1-12.0 u0026 micro; m.

TRANSWELL is placed in the culture plate, divided into two rooms: TRANSWELL small room, is called the upper chamber, which is placed on the upper culture fluid; the culture plate is referred to, and the lower culture liquid is placed. The upper and lower cultures are separated by a polycarbonate film.

The cell species is used in the upper chamber, and the ingredients in the lower culture solution can be investigated due to the polycarbonate film. The effect of cell growth, exercise, etc.
According to the characteristics of Transwell (aperture, membrane), the following aspects can be carried out: 1. Cultivate Aperture u0026 lt; 3.0 um Study the effect of the following cell B metabolite (secretion) on the upper chamber cell a.
2. Cell chemokination ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The amount of cells entering the room, reflecting the chemokinetic ability of the next chamber cells 3. Cell migration ( Aperture often selected 8.0, 12.0 u0026 micro; m to enter the lower cell volume of the next chamber, the reaction ability of the reaction in the chamber cell
4. Cell invasion The aperture often selects 8.0, 12.0 u0026 micro; M, the membranes have a matrix glue,
research into the lower cell volume of the next room, reflecting the room Cell invasion capabilities

Specific application example
Tumor cell invasion experiment (Transwell)
Principles:
The extracellular matrix in the body, the cells only secreted matrix metalloprotease (MMPs), and the degradation matrix gel can enter the room.

1. Transwell Chamber Preparation
1) No matrix glue Transwell Chamber Preparation
a. Package Base Film:
a. Matrigel (50mg / L) Press 1: 8 dilution, serum-free medium as dilution
b. Apply an appropriate amount to the Transwell chamber (24-well plate Transwell chamber plus 100 UL), 4 ° C operation.
C. In the 37 ° C incubator, incubate 4-5h ( u0026 gt; 5h); appears u0026 ldquo; white layer u0026 rdquo, indicating that it has become solid state.
2) Preparation
a. Small chamber is placed in the culture plate, and 300 u0026 micro; L-pre-temperature serum-free medium
b. Stand at room temperature 15-30min, so that the matrix glue is then hydrated
c. Remove the remaining culture fluid.

2. Cell suspension preparation
1) digestion, collect cells;
2) PBS wash 1-2 times
3) Non-serum-free medium retained (cell density at 1-10 u0026 Times; 105) / ml).
3. Cell vaccination
1) Take a proper amount of cell suspension to add the Transwell chamber (pressing Transwell). 24-well plate chamber general 200 u0026 micro; l.
2) The 24-well plate is generally added to the 500 u0026 micro; l-containing medium containing FBS or chemokines. Note that avoiding bubbles (lower culture fluids and small versions)
3) cultured cells: conventional cultivation 12-48h (mainly based on cancer cell invasion).
4. Results Statistics
There are two methods for detecting the number of cells passing through:
1) Direct counting method
u0026 ldquo; adhesive u0026 rdquo; Cell count:
u0026 nbsp u0026 ldquo; Nipped u0026 rdquo; refers to the cells after passing through the film, can be attached to the lower room side of the membrane without falling to the lower chamber. By staining the cells, cells can be counted under lens
a. Wipe the matrix glue and the cells in the upper chamber with cotton swabs
b. Dye: Commonly used 0.1% crystalline purple color.
Advantages: (1) Do not need to fix cells, direct dyeing.
(2) It is simple and convenient to formulate.
(3) 33% acetic acid can be used to measure the eluate OD570 valueIndirect reflection of the number of cells
Note that the membrane must be ventilated before dyeing, otherwise it may be dyed.
c. Cell count:
(1) Microscope to observe and take pictures
(2) Take 3-5 field counting cells

2) Indirect count method [123 ] For excessive cells, it is not possible to obtain accurate cell number by counting.
MTT method
a. Cotton swab rubbing the matrix glue and the cells in the upper chamber
b. 24-well plates to add 500 u0026 micro; l contain 0.5 mg / ml MTT medium, immerse the chamber, place Take it after 2-4 hours at 37 ° C.
c. The 500 u0026 micro; L DMSO is added to the 24-well plate, and the chamber is immersed in it, and the oscillation crystals are fully dissolved.
d. Remove the chamber to measure the OD value of the resulting DMSO.
Crystalline purple detection
a. Cotton swab rubbed the matrix and the cells in the upper chamber
b. (Optional) formaldehyde fixed for 30 minutes, room temperature
b. 24-well plates to add 500 u0026 micro; l The 0.1% crystalline hydrolysis was submerged in which the chamber was immersed in the room temperature for 20 minutes
c. The 24-well plate was added to 500 u0026 micro; L 33% acetic acid, and the chamber was immersed in the chamber.
d. Remove the chamber and measure the eluent OD570 value.
Advantages: The process of dyeing and decolorization does not affect the membrane cells, which can also be re-stained after decolorization.
5. Note
1TRANSWELL chamber:
Note whether it is pre-paved
2 upper culture fluid:
upper culture solution uses no serum medium for maintenance Osmotic pressure, generally added 0.05% -0.2% BSA.
3 Cells:
Cells with invasive ability can be used in Transwell invasion experiments.
4 matrix:
Commonly used artificial reconstructed base film material Matrigel
5 lower culture fluid:
Under the lower layer often contains medium containing 5% -10% FBS, the specific concentration is based on cell invasion The invasive cells can increase the FBS concentration.
6 Cell Crops:
Cell culture plates should be equipped with the purchased Transwell chamber.
III, Tumor Cell migration experiment (Transwell)
The process is basically the basics of Transwell invasion experiments. Different are not needed.
Step
1.Transwell placed in a 24-well plate with 600 ul of medium (containing 10% serum) in advance,
2. Add cells in the intercontope of Transwell (100 ul, diluted with 0.1% serum medium to the desired density),
3. Put the cell incubator and take out after culture time.
4. Remove Transwell, wipe the cells (interior side) of the film (side) by cotton swab
5. The other surface of the cell is fixed for 30 minutes with formaldehyde
6. Crystalline purple dyeing for 20 minutes,
7. PBS or water was washed 3,
8. Observations under microscope, the cells were observed.



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