Preparation method of flow cytometry sample for lavender and cell lysis buffer

A technology of flow cytometry and lysis buffer, applied in the field of cell lysis buffer, it can solve the problems of unsatisfactory results and complex genetic background, and achieve the effect of protecting DNA from being degraded, improving accuracy and reducing negative effects

Inactive Publication Date: 2017-11-24
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to its rich varieties, wide distribution, various shapes, and complex genetic background, some varieties of lavender cannot get ideal results when treated with conventional cell lysis buffer. Therefore, it is necessary to improve the special morphological structure and physiological characteristics of lavender. Cell lysis buffer, the final prepared sample is suitable for analysis by flow cytometry

Method used

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  • Preparation method of flow cytometry sample for lavender and cell lysis buffer

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Embodiment 1

[0055] In this embodiment, French lavender (Lavandula stoechas) is used as a processing sample, and the preparation method of a flow cytometry sample suitable for lavender provided in this embodiment is described. The preparation method includes the following steps:

[0056] 1 Take 1g of French lavender leaves, put them into a 5.5cm petri dish dripped with 1ml of pre-cooled cell lysis buffer, cut the lavender leaves into pieces within 1min with a blade;

[0057] Wherein, the component of cell lysis buffer is:

[0058] 0.2mol / L Tris-HCl, 10mmol / L MgCl 2 , 2mmol / L EDTA-2Na·2H 2 O, 80mmol / L NaCl, 10mmol / L Na 2 S 2 o 5 , 2% (w / v) PVP-10, 1% (V / V) Triton X-100, 5mmol / L DTT; pH value is 7.5.

[0059] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0060] 3 Filter the cell nucleus suspension with a 400-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plastic centrifuge tube;

[0061] 4 ...

Embodiment 2

[0066] The preparation method that the present embodiment is applicable to the flow cytometry sample of lavender, it comprises the steps:

[0067] 1 Take 0.2g of lavender leaves, put them into a petri dish dripped with 1ml of pre-cooled cell lysis buffer, cut the lavender leaves into pieces within 1min with a blade;

[0068] Wherein, the component of cell lysis buffer is:

[0069] 0.12mol / L Tris-HCl, 5mmol / L MgCl 2 , 3mmol / L EDTA-2Na·2H 2 O, 72mmol / L NaCl, 19mmol / L Na 2 S 2 o 5 , 1.5% (w / v) PVP-10, 0.7% (V / V) Triton X-100, 4mmol / L DTT; pH value is 7.5.

[0070] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0071] 3 Filter the cell nucleus suspension with a 400-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plastic centrifuge tube;

[0072] 4 Centrifuge the cell nucleus filtrate at 2000r / min at 4°C for 4 minutes, discard the supernatant and keep the precipitate, which is the c...

Embodiment 3

[0077] The preparation method that the present embodiment is applicable to the flow cytometry sample of lavender, it comprises the steps:

[0078] 1 Take 1g of lavender leaves, put them into a petri dish dripped with 1ml of pre-cooled cell lysis buffer, cut the lavender leaves into pieces within 1min with a blade;

[0079] Wherein, the component of cell lysis buffer is:

[0080] 0.16mol / L Tris-HCl, 14mmol / L MgCl 2 , 2mmol / L EDTA-2Na·2H 2 O, 86mmol / L NaCl, 14mmol / L NaCl 2 S 2 o 5 , 2.8% (w / v) PVP-10, 0.5% (V / V) Triton X-100, 6mmol / L DTT; pH value is 7.5.

[0081] 2 Add 1ml of pre-cooled cell lysis buffer to the fragments to release the nuclei and obtain a nuclei suspension;

[0082] 3 Filter the cell nucleus suspension with a 400-mesh nylon mesh to obtain the cell nucleus filtrate into a disposable 2ml plastic centrifuge tube;

[0083] 4 Centrifuge the cell nucleus filtrate at 4°C at 700r / min for 10 minutes, discard the supernatant and keep the precipitate, which is the ce...

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Abstract

The invention discloses a preparation method of a flow cytometry sample for lavender and a cell lysis buffer, and relates to the field of flow cytometry detection. The preparation method of the flow cytometry sample for lavender comprises the following steps: lysis: carrying out lysis treatment, filtration treatment and centrifuging treatment on a lavender tissue sample to obtain nucleus to be stained; and staining: carrying out staining treatment on the nucleus to be stained so as to obtain stained nucleus. According to the preparation method, an improved cell lysis buffer is used for treatment of the lavender tissue sample. The cell lysis buffer can remove residual cytoplasm fragments in the complete nucleus of lavender, maintain stability of nucleus in a suspension and prevent aggregation, protect DNA from being degraded, provide an appropriate environment for specific nuclear DNA chemical staining, and effectively reduce negative effect of cytoplast component on staining.

Description

technical field [0001] The invention relates to the field of flow cytometry detection, in particular to a method for preparing a flow cytometry sample suitable for lavender and a cell lysis buffer. Background technique [0002] Lavender (Lavandula angustifolia Mill) is a perennial subshrub of Labiatae, native to the Mediterranean coast. At present, there are 37 species and more than 100 varieties in the world. It is an important economic crop. Lavender has high ornamental value, and is widely used in medicine, chemical industry, health care and other industries. Now it is planted in many provinces of our country and has a large number of product development. Determining the genome size of some lavender varieties with important research value and economic traits can provide valuable reference for future lavender genome sequencing, research on its origin and evolution, and cross-breeding work. The understanding of chromosomal ploidy in lavender cells is of great significance ...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/30
CPCG01N1/28G01N1/30
Inventor蒋玮唐雪明吕贝贝吴潇
OwnerSHANGHAI ACAD OF AGRI SCI