Mycoplasma staining test kit use precautions - Database & Sql Blog Articles

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Mycoplasma staining test kit

Product introduction:

Mycoplasma staining detection reagent (MycoplasmaStainAssayKit) is a classic kit for detecting mycoplasma contamination by DNA fluorescent staining. The principle is that when cells undergo apoptosis, chromatin will shrink.

Hoechst33258

After staining, the nucleus of normal cells showed a normal blue color, while the nucleus of apoptotic cells showed dense dense staining, or densely stained in blocks, and the color was somewhat white.

LeageneHoechstStainingKit is often used for the detection of apoptosis in cultured iron or suspension cells as well as tissue sections. The kit detects a cell content ranging generally between 0.1 and 1 x 106.

product composition:

Name/number

RY0285-100T

Storage

Reagent A: Hoechst fixative

50ml

RT

Reagent B: Hoechst staining solution

50ml

-20

°C protected from light

Reagent C: Fluorescent sealing tablets

5ml

4

°C

Protected from light

user's Guide

1 copy

Bring your own materials:

1,

Fluorescence microscope or laser confocal microscope for observing blue light

2,

PBS or saline

3,

Slides, coverslips

4,

Pre-cooled fixative: pre-cooled 70% ethanol or 4% paraformaldehyde

Operation steps (for reference only):

(a) adherent cells

1. Take a clean coverslip soaked in 70% ethanol for 5 minutes or longer, dry it in a sterile clean bench or wash it 3 times with sterile PBS or saline, and wash it once with cell culture solution. The coverslips were placed in a 6-well plate or other culture dish, and the cells were seeded overnight to make the fusion rate about 50% to 80%.

2. After adding the intervention conditions to cause apoptosis of the cells, the culture solution is aspirated, and 0.5 ml of Hoechst fixative is added and fixed for 10 minutes or longer (4

°C overnight

).

3, remove the fixative, wash twice with PBS or saline, each time 3min, drain the liquid, wash with a shaker or manually shake.

4

Add 0.5 ml of Hoechst 33258 staining solution and incubate for 5 min. It is also advisable to use a shaker or shake it several times manually.

5. Discard the staining solution, wash it twice with PBS or normal saline for 3 minutes each time, drain the liquid, and shake it with a shaker or manually.

6. Drop a drop of anti-fluorescent seal on the slide, cover the cell cover with the cell cover, and let the cells contact the seal to avoid air bubbles.

7. Fluorescence microscopy can detect blue nuclei. The excitation wavelength is about 350 nm, and the emission wavelength is about 460 nm.

(two)

(two)

Suspension cells

1 Centrifugation to collect cell samples in a 1.5 ml centrifuge tube

,plus

Into Hoechst fixative 0.5ml, slowly suspend the cells, fixed for 10min or longer (also 4 ° C overnight).

2 Remove the fixative by low-speed centrifugation, wash twice with PBS or normal saline for 3 min each time, and drain the liquid. Shake it by hand several times during washing.

3 After low-speed centrifugation, remove most of the liquid and retain 50 ul of liquid. Slowly suspend the cells and add them to the slides to make the cells evenly distributed.

4 Dry slightly, so that the cells are attached to the slides and are not easy to flow with the liquid.

5

Evenly drip

Hoechst33258 staining solution 0.5ml, incubate for 5 minutes. Use absorbent paper to absorb liquid from the edge and dry slightly.

6 Discard the staining solution, wash it twice with PBS or normal saline for 3 minutes each time, and drain the liquid. Wash it with a shaker or shake it manually.

7 drops of anti-fluorescent seal on the slide, cover with a clean cover slip to avoid air bubbles.

8 fluorescence microscopy can detect blue nuclei. The excitation wavelength is about 350nm, and the emission wavelength is about 460nm.

(three)

Tissue sections

1 conventional embedded slice

.

2 Wash twice with PBS or normal saline for 3 min each time. Shake several times during washing.

.

3 evenly drop 0.5 ml of Hoechst 33258 staining solution and incubate for 5 minutes.

.

4 Discard the staining solution, wash it twice with PBS or normal saline. 3 minutes each time, drain the liquid. Use a shaker or shake manually.

.

5 Place the slice on the slide, drop a drop of anti-fluorescent seal, cover with a clean cover slip, and avoid bubbles as much as possible.

.

6 fluorescence microscopy can detect blue nuclei. Excitation wavelength is about 350nm

,

The emission wavelength is around 460nm

.

Precautions:

1

,

There is a problem of quenching in fluorescent dyes. It is recommended to detect them as soon as possible after dyeing, and should also be protected from light when using anti-fluorescent tablets.

.

2

,

In the centrifugation process for obtaining cell pellets, if the cell is not sufficiently precipitated for a particular cell, the centrifugal force or the centrifugation time may be appropriately increased.

.

3

,

Hoechst33258 staining solution is irritating to the human body, please pay attention to proper protection

.

4

,

For your safety and health,

Please wear

Lab coat with disposable gloves

.

Validity period:

Valid for 12 months

.

Can also be stored at 4 degrees Celsius, valid for one month

.