NAD +老化、癌症、神經退化性和代謝紊亂研究與分析

NAD +是一種二核苷酸輔助因子，具有在各種細胞還原氧化 (氧化還原) 反應中接受電子的潛力。還原形式的 NADH 是一種普遍存在的細胞電子供體。NAD +、NADH 和 NAD + /NADH 比率長期以來一直被認為可以控制多種氧化還原酶的活性。最近，已經鑑定出直接參與氧化還原控制的酶之外的酶能夠感知這些二核苷酸，包括NAD +依賴性蛋白脫酰酶的sirtuin家族。煙醯胺腺嘌呤二核苷酸 (NAD + ) 是每個活細胞中存在的關鍵輔酶，參與與細胞生物能相關的無數代謝過程。因此，NAD +經常在老化、癌症、神經退化性和代謝紊亂的背景下進行研究。

NAD+/NADH 比色測定試劑盒 | Abcam 貨號(ab282929)

NAD+/NADH Colorimetric Assay Kit (ab282929)

NAD+/NADH 比色測定試劑盒 (ab282929) 為靈敏檢測 NADH、NAD 及其比率提供了便利的工具。無需從樣品中純化 NADH 或 NAD。 NADH 選擇性顯色劑在與 NADH 反應時顯色，可在 OD 450 nm 處檢測。透過與提供的 NADH 標準品進行比較，可以輕鬆量化總 NAD。此方法定量、快速、簡單、靈敏，專為高通量格式而設計。此試劑盒可在 384 孔檢測板中檢測低至 0.04 pmol 的 NADH。此反應特異性檢測 NADH 和 NAD，但不檢測 NADP 或 NADPH。菸鹼醯胺腺嘌呤二核苷酸（Nicotinamide Adenine Dinucleotide, NAD）是多種催化蛋白活性不可或缺的輔酶。 NAD+ 和 NADH 及其還原形式參與許多生物氧化還原反應。 NAD+ 主要與分解代謝反應和能量產生所需的氧化還原酶有關。

Sirtuin 活性檢測試劑盒  | SIRT1 活性檢測試劑盒 | NMN | NAD+/NADH 比色測定試劑盒 | 細胞衰老檢測試劑盒 | Metformin | 雷帕黴菌（Rapamycin）

• Product name

  NAD+/NADH Colorimetric Assay Kit
  See all NAD/NADH kits

• Detection method

  Colorimetric

• Sample type

  Serum, Plasma, Tissue, Adherent cells, Suspension cells

• Assay type

  Quantitative

• Species reactivity

  Reacts with: Mammals

• Product overview

  This NAD+/NADH Colorimetric Assay Kit (ab282929) provides a convenient tool for a sensitive detection of NADH, NAD and their ratio. There is no requirement to purify NADH or NAD from samples. The NADH selective developer develops color while reacting with NADH and can be detected at OD 450 nm. The total NAD can be easily quantified by comparing with the provided NADH standard. The method is quantitative, rapid, simple, sensitive, and designed for high throughput format. The kit can detect as low as 0.04 pmol of NADH in a 384 well assay plate. The reaction specifically detects NADH and NAD, but not NADP nor NADPH.

  Sample types: Animal tissues: liver, kidney etc, Cell culture: adherent or suspension cells and Biological Fluids: Serum and Urine

• Notes

  This product is manufactured by BioVision, an Abcam company and was previously called K958 EZScreen™ NAD+/NADH Colorimetric Assay Kit (384-well). K958-400 is the same size as the 400 test size of ab282929.

  Nicotinamide Adenine Dinucleotide (NAD) is a co-enzyme indispensible for the activity of multiple catalytic proteins. NAD+ and NADH, its reduced form, participate in many biological redox reactions. NAD+ is mostly associated with redox enzymes required for catabolic reaction and energy production.

Storage instructions

Components	Identifier	400 tests
NAD Cycling Buffer	NM	1 x 15ml
NAD Cycling Enzyme Mix	Green	1 vial
NADH Developer	Purple	1 vial
NADH Standard	Yellow	1 vial
NADH/NAD Extraction Buffer	NM	1 x 50ml
Stop Solution	Red	1 x 1.2ml

NAD（煙醯胺腺嘌呤二核苷酸）(Nicotinamide adenine dinucleotide, NAD) 是代謝氧化還原反應(metabolic redox reactions)中的輔酶、多種細胞訊號分子(cell signaling molecules)的前體以及蛋白質翻譯後修飾(protein posttranslational modifications)的底物。NAD (Nicotinamide adenine dinucleotide, NAD)是一種二核苷酸，由透過磷酸基連接的兩個核苷酸組成：一個核苷酸含有腺苷環，另一個含有菸鹼醯胺(nicotinamide)。在新陳代謝中，NAD (Nicotinamide adenine dinucleotide, NAD)參與氧化還原反應，將電子從一個反應傳遞到另一個反應。因此，輔酶在細胞中以兩種形式存在：NAD (Nicotinamide adenine dinucleotide, NAD)是一種氧化劑 – 它接受來自其他分子的電子並被還原，形成 NADH，然後可用作還原劑來提供電子。這些電子轉移反應是NAD (Nicotinamide adenine dinucleotide, NAD)的主要功能。然而，它也用於其他細胞過程，最值得注意的是在翻譯後修飾中添加或去除蛋白質化學基團的酶底物。

細胞衰老檢測試劑盒（β-半乳糖苷酶） | 貨號KTA3030 

Cell Senescence Kit (β-Galactosidase Assay)

Figure 6. Effect of the fractions of fermented turmeric milk extract on SA-β-gal activity in UVA-irradiated Hs68 cells. (A) Microscopy images of senescent Hs68 cells shown by SA-β-gal staining (400× magnification). (B) SA-β-gal activity was quantified by the percentage of SA-β-gal-positive cells in total Hs68 cells. Superscripts indicate significant differences in one-way analysis of variance (ANOVA) and Tukey’s multiple range test (p < 0.05). SA-β-gal: senescence-associated β-galactosidase. NAC: N-acetylcysteine group. Hex100: 100 ppm of hexane fraction of fermented turmeric milk extract. EA100: 100 ppm of ethyl acetate fraction of fermented turmeric milk extract. Bu100: 100 ppm of butanol fraction of fermented turmeric milk extract. WA: 100 ppm of water residue fraction of fermented turmeric milk extract.

細胞衰老(Cellular senescence)是正常細胞停止分裂(cell divide)的現象。衰老的細胞(senescent cells)不再能夠複製(replicate)，但它們仍保持代謝活性(metabolically active)，但因阻滯於G1期，失去了對有絲分裂原的反應能力和合成DNA的能力，不能進入S期。並且與衰老相關的β-半乳糖苷酶活性染色(senescence-associated beta-galactosidase activity)呈陽性，這被認為是細胞衰老的生物標記(biomarker)。細胞的衰老(Cellular senescence)可以通過激活癌基因(oncogenes)和細胞-細胞融合(cell-cell fusion)來實現，DNA對活性氧（reactive oxygen species , ROS）升高的反應會導致DNA損傷(DNA damage)，而不是僅僅依賴於細胞分裂的數目。在正常衰老過程(normal aging)中，組織中的衰老細胞數量顯著增加。

細胞是生物結構(biological structure)和功能(function)的基本單位，也是生物衰老(biological aging)的基本單位。細胞衰老(Cellular senescence)在形態上表現為細胞結構(cell structure)的退化，例如核膜凹陷(nuclear membrane depression)，最終導致核膜塌陷(nuclear membrane collapse)，染色質結構(chromatin structure)改變，超二倍體(hyperdiploid)和異常多倍體細胞(polyploid cells)數量增加。細胞膜的脆性(cell membrane fragility)增加。通透性(permeability)降低，膜受體(membrane receptors)的類型和數量以及對配體(ligands)的敏感性發生變化；脂褐素(lipofuscin)在細胞中積累，許多細胞器和細胞內結構發生變性。細胞衰老(Cell senescence)在生理上表現(physiologically manifested)為功能下降和新陳代謝低下，例如細胞週期停滯(cell cycle arrest)，細胞複製能力 (cell replication ability) 喪失，對促有絲分裂刺激(mitogenic stimulation)的反應性減弱以及對促凋亡因子(pro-apoptotic factors)的反應性改變。細胞內酶活性中心被氧化，酶活性(Enzyme activity)降低，蛋白質合成(protein synthesis)降低等。衰老細胞(senescent cells)不再複制，但它們仍保持代謝活性(metabolically active)，並且衰老相關的β-半乳糖苷酶活性(beta-galactosidase activity)呈陽性，這被認為是細胞衰老生物標記(biomarker of cellular senescence)。

Abbkine細胞衰老檢測試劑盒（Senescence β-Galactosidase Staining Kit, KTA3030）提供了基於衰老過程中pH值為6的衰老相關β-半乳糖苷酶（SA-β-Gal）活性上調的試劑，用於檢測衰老細胞(senescent cells)或組織(tissues)。細胞或組織的老化可以在普通的光學顯微鏡下觀察。 SA-β-Gal僅存在於衰老細胞(senescent cells)中，在衰老前，靜止，腫瘤或永生細胞中無法檢測到Senescence β-Galactosidase Staining Kit  provide reagents for detecting the senescent cells or tissues based on the up-regulation of senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6 during aging. The aging of cells or tissues can be observed under a common optical microscope. SA-β-Gal is present only in senescent cells and is undetectable in presenescent, quiescent, tumor or immortal cells

• Kit components
• 10×Fixation Buffer • 10×PBS • Reagent A • Reagent B • Reagent C • X-Gal solution
• Features & Benefits
• Simple and optimized procedure. • Fast and convenient. • Suitable for aging detection of cultured cells and tissue sections. • Cell senescence β-galactosidase staining kit only stains senescent cells, and does not stain pre-senescence cells (senescent cells), quiescent cells (static cells), immortal cells (immortal cells) or tumor cells.
• Storage instructions

Refer to list of materials supplied for storage conditions of individual components. Stable for at least 12 months at recommended temperature from date of shipment.

• Shipping

Gel pack with blue ice.

• Precautions

The product listed herein is for research use only and is not intended for use in human or clinical diagnosis. Suggested applications of our products are not recommendations to use our products in violation of any patent or as a license. We cannot be responsible for patent infringements or other violations that may occur with the use of this product.

衰老細胞β-半乳糖苷酶染色試劑盒 | Aging Cell β-galactosidase Staining Kit Servicebio貨號G1073-100T (1ml為一個反應單位)

產品介紹:

大多數正常細胞的分裂能力(division ability)是有限的。當它們不能分裂(divide)時，就會進入衰老狀態(senescence)，稱為細胞衰老(cell senescence)。細胞衰老(Cell senescence)是細胞控制其生長潛能(growth potential)的保證機制，一般指複製性衰老(replicative senescence)。正常細胞在分裂次數有限後停止分裂，並發生不可逆的生長停滯(irreversible growth arrest)。此時，細胞還活著，但細胞形態(cell morphology)和生理代謝活動(physiological metabolic activity)發生了顯著變化，通常表現為細胞體積變大和與衰老相關的β-半乳糖苷酶(β-galactosidase)激活。 β-半乳糖苷酶(β-galactosidase)是細胞溶酶體(cell lysosomes)中的一種水解酶(hydrolytic enzyme)。它通常在 pH 4.0 時有活性，但在衰老細胞(senescent cell)中它在 pH 6.0 時有活性。本試劑盒正是基於這一現象和原理對衰老組織或細胞進行染色，以對抗與衰老相關的β-半乳糖苷酶(β-galactosidase)活性水平的上調。具體反應原理是以X-Gal為底物，衰老細胞(senescent cell)特異性β-半乳糖苷酶催化底物生成藍色產物，在細胞質中表現為藍色沉澱物，在光照下可觀察到顯微鏡。按每個樣品染色液用量1 mL計算，該試劑盒可完成100個樣品的染色。

Fig.1 WI-38 cells were stained with β-galactosidase kit. The left picture shows senescence WI-38 cells without division and proliferation ability but still alive. After staining, the positive staining cells were more than 95%. The image on the right shows newly resuscitated WI-38 cells (early passage) with less than 3 passages, and no obvious positive cells after staining.

Component Number	Component	G1073
G1073-1	β-galactosidase staining fixation solution	100 mL
G1073-2	β-galactosidase staining solution A	100 mL
G1073-3	β-galactosidase stain B	1.2 mL
G1073-4	DMF	5 mL
G1073-5	X-Gal（powder）	100 mg

Assay Protocol

l Preparation of reagents

1. Prepare your own PBS buffer (G4202 recommended).
2. 100 mg X-Gal powder was fully dissolved and mixed with 5 mL DMF (dimethylformamide), and then divided into 1.5 mL clean centrifuge tubes, 0.5 mL for each tube, and stored at -20℃ away from light. Avoid repeated freezing and thawing.
3. Preparation of β-galactosidase staining solution according to the proportion in the table below. For cells cultured in 6-well plates, 1.0-1.5 mL of staining working solution is required per well, and for 12-well plates, 0.5-1.0 mL of staining working solution is required per well. The staining solution was prepared according to the sample size to avoid waste.

Component	Volume
β-galactosidase staining solution A	940 μL
β-galactosidase stain B	10 μL
X – Gal solution	50 μL
Total Volume	1 mL

l Staining procedure

1. For adherent cells

(1) The cultured cells (or cell crawling sheets) in 6-well plates were aspirated and the cell culture medium was removed, washed twice with PBS, and 1 mL β-galactosidase staining fixing solution was added, and the cells were fixed for 15 min at room temperature.

(2) The fixed solution was discarded, and the cells were washed with PBS for 3 times, 2 min each time.

(3) PBS was removed by suction with a pipette, and 1 mL of β-galactosidase staining working solution was added to each well and incubated at 37℃ for 2 h to overnight. Note: Do not incubate in carbon dioxide incubator at 37 ° C. During the staining period, the color development should be observed in time. If the expression of β-galactosidase in the sample is high, the staining can be completed within a few hours. If β-galactosidase expression was low, the incubation time should be extended appropriately, during which the 6-well plate should be sealed with plastic wrap or parafilm to prevent liquid evaporation from affecting the staining results.

(4) Under the ordinary light microscope, the staining solution was removed after the positive cells developed color. If nuclei need to be counterstained, add a small amount of nucleosinophilic red dye solution (G1035 is recommended) to the well plate to cover the cells and stain at room temperature for 3 min, remove the staining solution, and wash with PBS several times.

(5) 2 mL PBS was added to cover the cells and the staining was completed. The sample could be stored at 4℃ for 1 week. Or add 70% glycerol to cover the cells, 4℃ can be stored for a long time. If it is the cell climbing sheet, the climbing sheet can be fully dried, xylene transparent after dropping neutral gum seal sheet, can be stored for a long time.

2. For frozen sections

(1) Rewarm frozen sections at room temperature for 10 min. Circle the tissue with tissue strokes.

(2) A proper amount of β-galactosidase staining fixing solution was added to the tissue to completely cover the tissue, and the solution was fixed at room temperature for 20 min.

(3) The tissue sections were soaked and washed in PBS for 3 times, 5 min each time.

(4) The sections were placed in a wet box to avoid light, and an appropriate amount of β-galactosidase staining solution was added to the tissue to completely cover the tissue. The wet box was incubated at 37℃ and the color development was observed under a microscope every 2 h. If no color development was observed, the culture was continued until the senescent cells on the tissue showed color. If the sample is to be incubated overnight, a sufficient amount of β-galactosidase staining solution should be added to prevent the staining solution from evaporating and drying the tablets.

(5) After the tissue developed color, the staining solution was removed, and the sections were immersed in PBS and washed twice, and then immersed in pure water and washed twice.

(6) (optional) Add nuclear solid red dye solution (G1035 is recommended) for 3 min and wash for 3 times.

(7) The slices were dehydrated with absolute ethanol for 2 times, then transparent with xylene for 5 min each time, and then sealed with neutral gum drop.

3. Staining results

The cytoplasm of senescent cells is scattered blue.

Note:

1. X-Gal solution should be thawed and mixed completely at room temperature before use.
2. β-galactosidase staining solution A and B should be restored to room temperature in advance before use, and the prepared staining solution should be thoroughly mixed without precipitation before use.
3. The β-galactosidase staining reaction of senescent cells is dependent on specific pH conditions, so it cannot be incubated in a CO2 incubator for color development, otherwise it will affect the pH of the staining solution and lead to staining failure.
4. When preparing dyeing solution, please choose consumables made of polypropylene (PP) or glass instead of polystyrene (PS).
5. The color development should be observed several times during the 2 h-overnight color development period, too short a time may lead to negative results; too much time can lead to false positives. The chromogenic time is closely related to the amount of β-galactosidase contained in the sample itself.
6. Before preparing the staining solution, check the pH value of staining solution A. If it is not 6.0 (which may be changed due to storage conditions), adjust the pH value to 6.0 with HCl or NaOH before use.
7. β-galactosidase staining of tissue sections requires high preparation of samples, which should be stored at -80℃ and tested as soon as possible. Because β-galactosidase is very easy to inactivate, improper storage or too long of the sample may lead to enzyme inactivation, then no positive staining.
8. Please wear a lab coat and disposable gloves during operation