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SenescenceCellsHistochemicalStainingKit

By Jerry Cunningham,2014-04-11 13:28
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SenescenceCellsHistochemicalStainingKit

    Senescence Cells Histochemical Staining Kit

    SIGMA Catalog # CS0030

    Preparation Instructions

    1. Before use, thaw all kit components and mix thoroughly until the

    solutions are homogenous.

    2. Ultrapure water (17 MΩ‧cm or equivalent) water should be used to

    prepare the working solutions for this kit.

    3. X-gal Solution - Warm the X-gal Solution at 37 ?C for 1 hour. Warming

    this solution is very important to avoid formation of aggregates that

    could interfere with the visualization of the stained cells. 4. 1× Fixation Buffer - Dilute the Fixation Buffer 10-fold with ultrapure

    water. Use of 0.2 mm filtered water is recommended. After

    preparation the 1× Fixation Buffer should be stored at 20 ?C. Prepare

    10 ml for 6 tests in a 35 mm plate or one 6 well plate. 5. 1× PBS - Dilute the PBS 10-fold with 0.2 mm filtered ultrapure water.

    After preparation, the 1× PBS should be stored at 28 ?C. Prepare 35 ml

    for 6 tests in a 35 mm plate or one 6 well plate. It is possible to prepare

    larger volumes and store the 1× PBS at 28 ?C.

    6. Staining Mixture - (Prepare just prior to use),Mix the following for

    preparation of 10 ml of the Staining Mixture:

    1 ml of Staining Solution 10×

    125 ul of Reagent B

    125 ul of Reagent C

    0.25 ml of X-gal Solution

    8.50 ml of ultrapure water

    Filter the Staining Mixture using a 0.2 mm filter to ensure there are no

    aggregates in the solution.

    Procedure

1. Aspirate the growth medium from the cells.

    2. Wash the cells twice with 1 ml of 1× PBS per well/plate. Carefully

    remove the entire wash solution by aspiration, so the cells do not

    detach.

    3. Add 1.5 ml per well of 1× Fixation Buffer and incubate the plate for 67

    minutes at room temperature.

    4. During the fixation process prepare the Staining Mixture as described in

    the Preparation Instructions.

    5. Rinse the cells 3 times with 1 ml of 1×PBS per well/plate.

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