YIJI cytoskeleton (actin monomer; G-ACTIN) red fluorescent staining kit product manual

YIJI cytoskeleton (actin monomer; G-ACTIN) red fluorescent staining kit product manual

(Chinese Version)

The main purpose

YIJI cytoskeleton (actin monomer; G-ACTIN) red fluorescent staining reagent is a DNase I designed to detect cytoskeletal actin monomer distribution and local orientation changes using Texas Red labeled DNase I. The authority of the state and the classic technical approach. The technology has been carefully developed and successfully tested. It is mainly used for the detection of cytoskeletal assembly of various living cells (animal, human, plant, etc.). The product is strictly sterile, ready to use, sensitive to reaction, simple in operation and stable in performance.

technical background

Actin is a 42KD globulin that constitutes the microfilament of the cytoskeleton and the thin filament of the muscle contraction device, as well as the pseudopodia and microvilli on the cell membrane surface. Actin is present in all eukaryotic cells and is highly evolved and involved in a variety of important cellular functions including cell migration, cell division, muscle contraction, cell morphology maintenance, membrane trafficking, and the like. Mammals have six isomers, which are classified into three types: α, β, and γ. G-actin combines to grow silk aggregates under physiological conditions and transform into filamentous actin (F-actin) to form microfilaments of the cytoskeleton. The dynamic change in the polymerization and depolymerization of actin is one of the main parameters of cytoskeleton assembly. Bovine pancreatic DNase I has a high affinity binding to actin monomer (G-actin), so it can detect cell G-muscle in vitro by Texas Red-labeled DNase I. The distribution and content of actin, according to which the regulation mechanism of cytoskeletal assembly is analyzed.

product content

YIJI cleaning solution (Reagent A) ml

YIJI Fixative (Reagent B) ml

YIJI Translucent Liquid (Reagent C) ML

YIJI Blocker (Reagent D) ML

YIJI staining solution (Reagent E) microliter

YIJI counterstain solution (Reagent F) microliter

Product manual 1 copy

storage method

Save YIJI staining solution (Reagent E) and YIJI counterstaining solution (Reagent F) in the -20 °C refrigerator to avoid repeated freezing and thawing; the rest are stored in a 4 ° C refrigerator to avoid light; effective to ensure June

User-supplied

Trypsin ethylenediaminetetraacetic acid mixture (GMS12024): for cell detachment

Complete cell culture fluid (GMS12052): for the treatment of neutralizing trypsin

15 ml conical centrifuge tube: container for sample handling

1.5 ml centrifuge tube: container for cell staining

(Micro) Benchtop Centrifuge: for sample handling

Fluorescence (Confocal) Microscopy: for Cell Fluorescence Observation

Experimental procedure

Before the start of the experiment, the reagent in the -20 ° C refrigerator was allowed to freeze and thaw at room temperature. Transfer xx μl of YIJI permeate ( R e agent C ) to a 1.5 ml centrifuge tube, then add xx μl of YIJI staining solution ( Reagent E ) and YIJI counterstaining solution ( Reagent F ) , and mix them. The YIJI dyeing working solution is placed in an ice bath and placed in a dark room for later use. Then do the following.

• Direct method

1. Prepare a cell culture 6-well plate with 1 cell 1 X 1 cm size slide
2. Inoculate the cells to be tested until the confluence rate per well reaches 70%.
3. Carefully remove the cell culture fluid
4. Gently add xx μl of YIJI Cleaner ( Reagent A ) onto the cell slide to cover the sample surface.
5. Carefully remove the YIJI cleaning solution ( Reagent A )
6. Gently add xx l YIJI fixing solution (Reagent B) slides onto the cells covering the surface of the sample
7. Incubate in an ice bath for 30 minutes
8. CAUTION skipping YIJI fixing solution (Reagent B)
9. Gently add xx l YIJI transparent liquid (Reagent C) to slide on the cell, the sample surface covering the
10. Carefully remove YIJI Transparency Fluid ( Reagent C )
11. Gently add xx YIJI l blocking solution (Reagent D) to slide on the cell, the sample surface covering the
12. Incubate for 30 minutes at room temperature
13. Carefully remove the YIJI blocking solution ( Reagent D )
14. Gently add xx μl of YIJI Cleaner ( Reagent A ) onto the cell slide to cover the sample surface.
15. Carefully remove the YIJI cleaning solution ( Reagent A )
16. Repeat experiment steps 14 and 15 once
17. Carefully add xx microliters of YIJI staining solution to cover the sample surface
18. Incubate for 60 minutes at room temperature in the dark room
19. Carefully remove the YIJI dyeing solution
20. Gently add xx μl of YIJI Cleaner ( Reagent A ) onto the cell slide to cover the sample surface.
21. Carefully remove the YIJI cleaning solution ( Reagent A )
22. Cover
23. Immediately under fluorescence (confocal) microscopy (200 cells per field count): excitation wavelength 597 nm, emission wavelength 618 nm (G-actin staining) or excitation wavelength 350 nm, emission wavelength 460 nm (nuclear staining) -

Red fluorescence shows G-actin; blue fluorescence shows nuclei as rings or ovals

• Indirect method

1. Prepare a cell of 25 cm ² cell culture flask until the confluence rate reaches 70% (approximately 1 million cell numbers)
2. Carefully remove the cell culture fluid into a 15 ml conical centrifuge tube (collect the suspension cells)
3. Add 2 ml of YIJI cleaning solution ( Reagent A ) to cover the cell surface
4. Carefully remove 2 ml of YIJI Cleaner ( Reagent A ) into a 15 mL Conical Tube
5. Add 1 ml of user-prepared trypsin ethylenediaminetetraacetic acid mixture to cover the cell surface
6. Incubate in a 37 ° C incubator for 1 minute
7. Gently shake the vibrating flask to remove the cells
8. Add 3 ml of user-prepared complete cell culture medium
9. Move into a 15 ml conical centrifuge tube (note: suspension cells can start from this step)
10. Place in a benchtop centrifuge for 5 minutes at a speed of 300g
11. Carefully remove the supernatant
12. Add 500 μl of YIJI Cleaner ( Reagent A ) and mix
13. Transfer to a 1.5 ml centrifuge tube
14. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
15. Carefully remove the supernatant
16. Add 500 microliters YIJI fixing solution (Reagent B), mix
17. Incubate in an ice bath for 30 minutes
18. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
19. Carefully remove the supernatant
20. Add 500 microliters YIJI transparent liquid (Reagent C), mix
21. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
22. Carefully remove the supernatant
23. Add 500 microliters YIJI blocking solution (Reagent D), mix
24. Incubate for 30 minutes at room temperature
25. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
26. Carefully remove the supernatant
27. Add 500 μl of YIJI Cleaner ( Reagent A ) and mix
28. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
29. Carefully remove the supernatant
30. Add 100 μl of YIJI dyeing working solution
31. Gently pump up and down with a 200 μl pipette and mix the cell pellets
32. Incubate for 60 minutes at room temperature in the dark room
33. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
34. Carefully remove the supernatant
35. Add 500 μl of YIJI Cleaner ( Reagent A ) and mix
36. Place in a mini tabletop centrifuge for 5 minutes at a speed of 300g (or 1500RPM, eg EPPENDORF 5415)
37. Carefully remove the supernatant
38. Add 200 μl of YIJI Cleaner ( Reagent A ) and mix
39. Immediately remove 10 microliters and compress on a glass slide
40. Observation under a fluorescence (confocal) microscope (200 cells per field count): excitation wavelength 597 nm, emission wavelength 618 nm (G-actin staining) or excitation wavelength 350 nm, emission wavelength 460 nm (nuclear staining) -

Red fluorescence shows G-actin; blue fluorescence shows nuclei as rings or ovals

Precautions

1. This product is 10 operations
2. Wear gloves during the entire operation to prevent contamination
3. Light must be avoided when incubating
4. It is recommended to detect a cell volume of 10 ⁶ cells.
5. Users can adjust the amount of reagents according to the situation.
6. It is recommended that the fluorescence analysis be performed immediately after the cell staining is completed. It should not exceed 1 hour.
7. The company provides a series of cytoskeletal detection reagent products

Quality Standard

1. This product has been certified to be stable.
2. This product has been identified as fluorescent