- 背景資料
細胞存活率試驗(MTT 試驗)(MTT assay)，是一種藉由測量活體細胞數量的吸光值，以此來計算出細胞的存活率的方法。MTT為一種黃色化合物，其全名為(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)，當MTT進入活體細胞的粒線體時，會發生氧化還原的作用，MTT會被還原成甲䐶(formazan)，甲䐶為一種紫藍色化合物，可被DMSO溶解，並可在分光光度計(spectrophotometer) 570 nm的波長下顯色，以此測量其吸光值。
- A. MANUAL ASSAY PROCEDURE:
- B. AUTO-ANALYSER ASSAY PROCEDURE:
- C. MICROPLATE ASSAY PROCEDURE:
- SAMPLE PREPARATION:
1. Sample dilution.
The amount of L-ascorbic acid present in the cuvette (i.e. in the 0.1 mL of sample being analysed) should range between 0.5 and 30 μg. The sample solution must therefore be diluted sufficiently to yield an L-ascorbic acid concentration between 0.005 and 0.30 g/L.
2. Sample clarification.
The Carrez-clarification protocol cannot be used in sample preparation for L-ascorbic acid determination due to the resultant low recovery of the analyte.
In place of Carrez-reagents, samples are clarified and protein is precipitated and removed by a combination of treatment with 3% (w/v) metaphosphoric acid plus 10 mM EDTA buffer and filtration. For the preparation of 3% (w/v) metaphosphoric acid plus 10 mM EDTA buffer, see page 4.
3. General considerations.
(a) Liquid samples: clear, slightly coloured liquid samples can be used directly in the assay.
(b) Alkaline samples: if an alkaline sample is to be used undiluted, the pH of the solution should be adjusted to approx. 3.5 using 2 M HCl.
(c) Carbon dioxide: samples containing a significant amount of carbon dioxide, such as beer, should be degassed by filtration or by stirring with a glass rod.
(d) Coloured samples: lightly coloured samples can be analysed directly.
(e) Strongly coloured samples: if used undiluted, strongly coloured samples should be treated by the addition of 0.2 g of polyvinylpolypyrrolidone (PVPP)/10 mL of sample. Shake the tube vigorously for 5 min and then filter through Whatman No. 1 filter paper.
(f) Solid samples: homogenise or crush solid samples in 3% (w/v) metaphosphoric acid plus 10 mM EDTA and filter if necessary.
(g) Samples containing fat: extract such samples with 3% (w/v) metaphosphoric acid plus 10 mM EDTA at a temperature above the melting point of the fat, e.g. in a 100 mL volumetric flask at 60°C. Cool to allow the fat to separate and make up to the mark with 3% (w/v) metaphosphoric acid plus 10 mM EDTA. Filter the solution, discard the first few mL of filtrate and use the clear supernatant (which may be slightly opalescent) for the assay.
(h) Samples containing protein: deproteinise samples containing protein by adding an equal volume 1 M potassium phosphate buffer (pH 3.5) with mixing. Centrifuge at 1,500 g for 10 min and dilute with 3% (w/v) metaphosphoric acid plus 10 mM EDTA.
商品特色
商品規格
- 商品規格
40 assays (manual) / 400 assays (microplate) / 400 assays (auto-analyser)
Bottle 1:
Buffer (44 mL, pH 5.6).
Stable for > 2 years at 4°C.
Bottle 2:
MTT (18 mL, pH 3.5).
Stable for > 2 years in the dark at room temperature.
Bottle 3: *2
PMS.
Stable for > 2 years in the dark at 4°C.
Bottle 4:
Ascorbic acid oxidase suspension (0.85 mL).
Stable for > 2 years at 4°C.
Bottle 5:
L-Ascorbic acid (vitamin C) (~ 2 g).
Stable for > 2 years; store sealed at 4°C.
