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Preparation method of marine invertebrate blood cell suspension

A technology for invertebrates and blood cells, applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve problems such as the absence of calcium and magnesium ions, and achieve the effects of simple operation, small damage, and high cell activity

Active Publication Date: 2022-02-11
BEIBU GULF UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, single-cell sequencing technology needs to solve three problems: first, the sample must be a suspension of a single cell; second, the number of living cells in the sample must reach more than 80%; third, the solution for making the cell suspension must not contain calcium and magnesium ions exist
However, the preparation method of the cell suspension reported so far cannot satisfy the above three conditions at the same time.

Method used

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  • Preparation method of marine invertebrate blood cell suspension
  • Preparation method of marine invertebrate blood cell suspension
  • Preparation method of marine invertebrate blood cell suspension

Examples

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Comparison scheme
Effect test

Embodiment 1

[0030] A preparation method of marine invertebrate blood cell suspension, comprising the steps of:

[0031] 1) Use a 1mL syringe to extract 3mL of blood from the adductor muscle of the Hepu pinnacle shell, add the improved anticoagulant, and add the improved anticoagulant to the blood at a volume ratio of 3:1; Dissolve 4.5g of EDTA, 9.0g of sodium citrate, 25.0g of glucose, and 24.0g of NaCl in deionized water, adjust the pH to 8.0, and filter and sterilize with a 0.22μm filter membrane;

[0032] 2) After mixing, filter with a 40 μm cell sieve; the filtrate was centrifuged at 4°C and 400 g for 6 min to recover the cells;

[0033] 3) Discard the supernatant, and add 1 mL of pre-cooled balanced salt solution to the precipitate;

[0034] 4) Gently pipet 10 times with a wide-mouthed pipette tip, resuspend the cells, and centrifuge at 4°C for 6 minutes at 400g to recover the cells; discard the supernatant, add 5 mL of pre-cooled balanced salt solution to the pellet, and use a wide...

Embodiment 2

[0045] A preparation method of marine invertebrate blood cell suspension, comprising the steps of:

[0046] 1) Use a 1mL syringe to extract 3mL of blood from the adductor muscle of the abalone, add the improved anticoagulant, and add the improved anticoagulant to the blood at a volume ratio of 2:1; the improved anticoagulant is dissolved in every liter of deionized water 4.0g of EDTA, 8.3g of sodium citrate, 22.0g of glucose, 21.0g of NaCl, adjust the pH to 7.5, and filter and sterilize with a 0.22μm filter membrane;

[0047] 2) After mixing, filter with a 40 μm cell sieve; the filtrate was centrifuged at 4°C and 400g for 8 minutes to recover the cells;

[0048] 3) Discard the supernatant, and add 1 mL of pre-cooled balanced salt solution to the precipitate;

[0049] 4) Use a wide-mouthed pipette tip to blow gently 5 times, resuspend the cells, and centrifuge at 4°C, 400g for 8 minutes to recover the cells; discard the supernatant, add 5 mL of pre-cooled balanced salt solutio...

Embodiment 3

[0060] A preparation method of marine invertebrate blood cell suspension, comprising the steps of:

[0061] 1) Use a 1mL syringe to extract 3mL of blood from the clam adductor muscle, add the improved anticoagulant, and add the improved anticoagulant to the blood at a volume ratio of 2:1; the improved anticoagulant is dissolved in every liter of deionized water 4.5g of EDTA, 9.0g of sodium citrate, 25.0g of glucose, 24.0g of NaCl, adjust the pH to 8.0, and filter and sterilize with a 0.22μm filter membrane;

[0062] 2) After mixing, filter with a 40 μm cell sieve; the filtrate was centrifuged at 4°C and 400g for 8 minutes to recover the cells;

[0063] 3) Discard the supernatant, and add 1 mL of pre-cooled balanced salt solution to the precipitate;

[0064] 4) Gently pipette 10 times with a wide-mouthed pipette tip, resuspend the cells, and centrifuge at 4°C, 400g for 10 minutes to recover the cells; discard the supernatant, add 5 mL of pre-cooled balanced salt solution to th...

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Abstract

The invention discloses a preparation method of a marine invertebrate blood cell suspension. The method simplifies the operation process, and maximally ensures the cell activity through the steps of uniformly mixing extracted blood cells with an anticoagulant, sieving, centrifuging, resuspending the cells with a balanced salt solution and the like, thereby obtaining the blood cell suspension with good cell state, high survival rate and few impurities. The method can be used for research on subsequent blood cell typing and functions of marine invertebrates.

Description

【Technical field】 [0001] The invention relates to the technical field of marine invertebrate cell biology, in particular to a preparation method of marine invertebrate blood cell suspension. 【Background technique】 [0002] Marine invertebrates are the main species of mariculture in my country, accounting for the highest proportion of marine organisms, with a wide variety of phylum and species, and high economic value. In particular, mollusks (shellfish) and crustaceans (shrimps and crabs) are the main breeding objects in my country's coastal areas. For marine invertebrates and higher animals, they lack a complete set of immune systems in their bodies and mainly rely on non-specific immune systems for body defense. The immune defense process is mainly composed of cellular immunity mediated by blood cells and hemolymph and immune Related enzyme-mediated humoral immunity to complete. Blood cells are the main executors of cellular immunity in marine invertebrates, but differen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/24
CPCC12Q1/24Y02A40/81
Inventor 白丽蓉王培喻达辉李素萍郑玉斯何积翠郭颖
Owner BEIBU GULF UNIV
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