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Pretreatment of cell samples

First,homogenate medium

        Generally, 0.05 mol/L Tris-HCl, pH 7.4 phosphate buffer (PBS) is used. The customer can set the concentration according to the sample and the measurement index, in order to maintain the isotonic environment of the sample.

Second,the pretreatment of the cell sample:

1. Collection of cell pellets:

    ① Suspension cells:For suspension cultured cells, the cell pellet was collected by centrifugation directly, and the culture solution was centrifuged at 1000 rpm for 10 minutes at room temperature, and the supernatant of the retained cells was discarded.

    ② Adherent cells:For adherent cultured cells, digest the cells with trypsin, or scrape the cells with cell scraping, centrifuge the culture solution at 1000 rpm for 10 minutes at room temperature, discard the supernatant cells. precipitation.

We generally advise customers that the cell density should not be less than one million / ml.

2. Washing of cell pellets:

        0.5-1 ml of PBS (isotonic) was added to the cell pellet, and the mixture was gently inverted. The culture was centrifuged at 1000 rpm for 10 minutes at room temperature, and the supernatant was discarded.

Repeat the above operation repeatedly for 1 to 2 times.

3, the way of homogenization: manual homogenization, ultrasonic disruption, lysate lysis, repeated freezing and thawing.

    ① Manual homogenization: Add a certain amount of PBS to the cell pellet (the amount of PBS added varies according to the measured index, generally 0.5ml, cell density is not less than 1 million / ml), mix, The cells were suspended in PBS, and the cell suspension was pipetted into a glass homogenate tube (2 ml glass homogenate tube), and the glass homogenate tube was placed in an ice-water mixture, manually homogenized for 3 minutes, and then taken. The disrupted cell suspension was assayed.

    ② ultrasonic fracture:Add a certain amount of PBS to the cell pellet (the amount of PBS added varies according to the measured index, generally 0.5ml, cell density is not less than 1 million / ml), mix, and suspend the cells in PBS. In the ice bath condition, proceed as follows:

     a, using an ultrasonic pulverizer for pulverization, sonicrep150 type ultrasonic generator can be used to ultrasonically process the amplitude of 14 micron for 30 seconds to break the cells, or use domestic ultrasonic generator, with 400 amps, 5 seconds / time, gap 10 seconds repeated 3 ~ 5 Times.

      b. Use ultrasonic cell crusher, 300W power, each ultrasound for 3 to 5 seconds, interval 30 seconds, repeat 4 to 5 times.

    ③lysate lysis:Commonly used lysates are SDS, NP-40, and TritonX-100. The effects of these three detergents are different, or the strength of the action is different.

    1. SDS is an ionic detergent. It is the most powerful. It can basically completely break the cells, the DNA will be released, and the lysate becomes very viscous.

    2, NP-40 is a very mild detergent, 1% concentration can basically destroy the membrane, but weak on the nuclear membrane damage, combined with a specific buffer can obtain cytoplasmic protein.

    3, TritonX-100's ability between NP40 and SDS, biased to NP40, is also one of the commonly used cell lysate components, has a role in the protection of protein activity (SDS will basically deactivate protein denaturation).

Detergent properties are only one aspect, buffer, ratio, concentration, extraction method and material handling are also key factors.

Since many lysates are protein denaturants, there is a certain influence on the determination of enzyme activity. It is generally not recommended to use lysates for lysis.

    ④ repeated freezing and thawing:Add a certain amount of PBS to the cell pellet (the amount of PBS added varies according to the measured index, generally 0.5ml, cell density is not less than 1 million / ml), mix, and suspend the cells in PBS. In the middle, put the ice in the freezer, dissolve, then freeze, and dissolve again, repeated about 3 times).

      Because repeated freeze-thaw has a great influence on enzyme activity, it is generally not recommended.