Cell Proliferation / Cell Cytotoxicity Assay
Cell viability and cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals. They are based on various cell functions such as enzyme activity, cell membrane permeability, cell adherence, ATP production, co-enzyme production, and nucleotide uptake activity. A widely used assay for staining dead cells is Trypan Blue. In this method, cell viability must be determined by counting the unstained cells with a microscope or other instruments.
However, Trypan Blue staining cannot be used to distinguish between healthy cells and cells that are alive but losing cell functions. Because of this, enzyme-based methods have been gaining more attention. Fig. 1 indicates various reagents used for detection of cell viability.
Among the enzyme-based assays, the MTT assay is the best known for determining mitochondrial dehydrogenase activities in living cells. MTT is reduced to a purple formazan by NADH. MTT formazan, however, has low solubility in water and it forms purple needle-shaped crystals in cells. Before measuring absorbance, an organic solvent is required to dissolve the crystals. Additionally, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to many floating cells with MTT formazan needles, giving significant well-to-well error.
Although the 3[H]thymidine-uptake assay is regarded as a reliable cell viability assay, the use of a radioisotope causes various concerns. The 51Cr method is highly sensitive and is commonly used to determine low levels of cytotoxicity.
However, the use of 51Cr also causes problems in handling, storage, and disposal of the material. Cellular enzymes such as lactate dehydrogenase (LDH), adenylate kinase, and glucose-6-phosphate dehydrogenase are also used as cell death markers, and there are several such products available on the market. However, adenylate kinase and glucose-6-phosphate are not stable; only lactate dehydrogenase does not lose its activity during cell death assays. Therefore, cell death assays based on LDH activity are more reliable than other enzyme-based cell death assays.
Dojindo developed highly water-soluble tetrazolium salts called WSTs. WSTs produce water-soluble formazans and are suitable for assays of cell proliferation and cytotoxicity. WSTs receive two electrons from viable cells to generate a yellow, orange, or purple formazan dye. WST-8, a highly stable WST, is utilized in Cell Counting Kit-8 (CCK-8). The electron mediator used in this kit, 1-Methoxy PMS, is also highly stable. Therefore, CCK-8 is stable for at least 6 months at room temperature and one year at 0-5°C. Since WST-8, WST-8 formazan, and 1-Methoxy PMS have no cytotoxicity in cell culture media, additional experiments may be carried out using the same assay plate. Dehydrogenase-based assays reflect cell conditions with more sensitivity than the other assays because they depend on several elements including dehydrogenases, NAD(H), NADP(H), and mitochondrial activity. The major difference between CCK-8 and the MTT assay, other than MTT’s toxicity, is the enzymes involved.
The CCK-8 assay involves most of the dehydrogenase in a cell, while MTT only involves mitochondrial dehydrogenase. Therefore, the MTT assay depends on mitochondrial activity, not the cell itself. Additionally, CCK-8 is far more sensitive than the MTT assay. Since WST-8 formazan is water soluble, it does not form crystals as MTT does. Therefore, after 1-4 hours of incubation with the CCK-8 solution, measurement of O.D. at 450 nm gives the number of viable cells. No extra steps are required.
Cell Death Assay: Cytotoxicity LDH Assay Kit-WST
Cytotoxicity LDH Assay Kit-WST is used for cytotoxicity determination by measuring the activity of lactate dehydrogenase (LDH) released. LDH is a stable cytoplasmic enzyme found in all types of cells and is released into the cell culture medium when the plasma membrane is damaged.
Esterase Activity Based Assay: Cell Counting Kit-F
Cell Counting Kit-F is another cell viability assay kit based on the esterase activity of viable cells. Fluorescent esterase substrates, such as Calcein-AM, BCECF-AM, CFSE, and FDA, can be passively loaded into viable cells. These electrically neutral molecules, which have acetyl or acetoxymethyl groups capping the phenolic OH or carboxylic groups of Calcein, freely permeate into the cell. Intracellular esterases then convert them into green fluorescent products, which are retained by cells. These esterase substrates, therefore, can serve as viability assay probes for cells. Calcein-AM is used for the kit because it is retained inside of cells better than the other fluorescent esterase substrates.
Dead and Viable Cell Staining: Double Staining Kit
Propidium iodide and Calcein-AM are used to distinguish dead and viable cells simultaneously. Dead cells can be stained by propidium iodide because it passes through deteriorated cell membranes; viable cells can be stained by Calcein-AM because of their esterase activity. Since both calcein and propidium iodide-DNA complex can be excited at 490 nm to emit green and red fluorescence, respectively, dead and viable cells can be observed with a fluorescence microscope. Only dead cells are observed with 545 nm excitation.
- Cell Proliferation / Cell Cytotoxicity Assay Kits
Code Product name Unit size CK17 Cell Proliferation / Cytotoxicity Assay Kit
Viability/Cytotoxicity Multiplex Assay Kit
500 tests UP04 Amino Acid Uptake Assay
Amino Acid Uptake Assay Kit
UP05 Cystine Uptake Assay Kit
Cystine Uptake Assay Kit
UP07 Fatty Acid Uptake Assay
Fatty Acid Uptake Assay Kit
- Related Reagents
Utafiti wa Cell viability na cytotoxicity hutumiwa kwa ajili ya uchunguzi wa madawa ya kulevya na vipimo vya cytotoxicity ya kemikali. Hizi hutegemea kazi mbalimbali za chembe kama vile shughuli za enzyme, membrane ya chembe, uaminifu wa chembe, uzalishaji wa ATP, uzalishaji wa coenzyme, na shughuli ya uingizaji wa nucleotide. Kiwango kinachotumiwa sana kwa ajili ya kupaka rangi chembe zilizokufa ni Trypan Blue. Katika mbinu hii, viability ya chembe lazima kuamua kwa kuhesabu chembe unstained na darubini au vyombo vingine. Hata hivyo, rangi ya Trypan Blue haiwezi kutumiwa kutofautisha kati ya chembe zenye afya na chembe zilizo hai lakini zinazopoteza kazi za chembe. Kwa sababu hiyo, mbinu zinazotumiwa na vimeng'enya zimekuwa zikipata uangalifu zaidi. Mchoro. 1 inaonyesha reagents mbalimbali kutumika kwa ajili ya kugundua viability ya chembe. Mtihani wa MTT ni moja ya vipimo vya msingi wa enzyme, na ni muhimu kwa kuamua utendaji wa mitochondrial dehydrogenase katika seli za hai. MTT hupunguzwa kuwa formazan ya zambarau na NADH. MTT-formase ina uwezo mdogo wa kuyeyuka katika maji na huunda fuwele za rangi ya zambarau katika chembe. Kabla ya kupima absorbance, solvent kikaboni inahitajika kuyeyusha fuwele. Kwa kuongezea, cytotoxicity ya MTT-formase inafanya kuwa vigumu kuondoa vyombo vya habari vya utamaduni wa seli kutoka kwa visima vya sahani kwa sababu ya seli nyingi zinazoelea na sindano za MTT-formase, ikitoa kosa kubwa la vizuri. Ingawa 3H-thymidine-uptaking assay ni kuchukuliwa kama uhakika seli viability assay, matumizi ya radioisotope husababisha wasiwasi mbalimbali. Njia ya 51Cr ni nyeti sana na hutumiwa kwa kawaida kuamua viwango vya chini vya cytotoxicity. Kwa sababu ya matumizi ya 51Cr, ni muhimu kuondoa, kuhifadhi, na kuondoa vitu vya kemikali. Enzymes za seli kama vile lactate dehydrogenase (LDH), adenylate kinase, na glucose-6-phosphate dehydrogenase pia hutumiwa kama alama za kifo cha seli, na kuna bidhaa kadhaa kama hizo zinazopatikana sokoni. Hata hivyo, adenylate kinase na glucose-6-phosphate si imara; tu lactate dehydrogenase haina kupoteza shughuli yake wakati wa vipimo vya kifo cha seli. Kwa hiyo, vipimo vya kifo cha seli kulingana na shughuli ya LDH ni ya kuaminika zaidi kuliko vipimo vingine vya kifo cha seli kulingana na enzyme. Dojindo aliendeleza chumvi za tetrazolium zinazoweza kuyeyuka vizuri katika maji zinazoitwa WSTs. WSTs kuzalisha formazans maji mumunyifu na ni sahihi kwa ajili ya vipimo ya kuongezeka kwa seli na cytotoxicity. WST hupokea elektroni mbili kutoka kwa chembe zenye uwezo wa kuishi ili kutokeza rangi ya manjano, machungwa, au zambarau ya formazan. WST-8 ni kitambaa cha WST-K ambacho kina uwezo wa kuunganisha chembe za urithi na chembe za urithi. Msaidizi wa elektroniki anayetumiwa katika kit hii, 1-Methoxy-PMS, pia ni thabiti sana. Kwa hiyo, CCK-8 ni imara kwa angalau miezi sita katika joto la chumba na mwaka mmoja katika joto la 0-5 ° C. Kwa kuwa WST-8 na WST-8-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P- Kwa mfano, vipimo vya dehydrogenase huonyesha hali ya seli kwa unyeti zaidi kuliko vipimo vingine, kwa sababu zinategemea vipimo kadhaa, ikiwa ni pamoja na dehydrogenase, NAD +, NAD +, NAD +, na shughuli ya mitochondrial. Tofauti kuu kati ya CCK-8 na kipimo cha MTT, mbali na sumu ya MTT, ni enzymes zinazohusika. Kwa mfano, mtihani wa CK8 unajumuisha dehydrogenase ya mitochondrial, na mtihani wa MTT unajumuisha dehydrogenase ya mitochondrial. Kwa hiyo, kipimo cha MTT hutegemea utendaji wa mitochondrial, si chembe yenyewe. Kwa kuongezea, CCK-8 ni nyeti zaidi kuliko kipimo cha MTT. Kwa kuwa WST-8 formazan huyeyuka katika maji, haifanyi fuwele kama MTT. Baada ya masaa 1 hadi 4 ya incubation na suluhisho la CKK-8, kipimo cha ODD kinapaswa kuchukuliwa. Katika 450 nm, idadi ya chembe za viable ni: Hakuna hatua za ziada zinazohitajika. LDH-WST (Cytotoxicity Assay Kit) ni kitambulisho cha LDH-WST (Lactate dehydrogenase) LDH ni enzyme ya cytoplasmic imara inayopatikana katika aina zote za seli na hutolewa katika mazingira ya kukuza seli wakati utando wa plasma umeharibiwa. Kiwango cha Esterase Activity Based Assay (ECAS) - Kit-F ya Hesabu ya Chembe (Cell Counting Kit-F) ni kit-F ya hesabu ya viability ya chembe kulingana na utendaji wa esterase ya chembe zinazoweza kuishi. Substrate ya fluorescent esterase, kama vile Calcinein-AM, BCF-AM, CFSE, na FDA, inaweza kubeba passively katika seli hai. Molekuli hizi za umeme zisizo na umeme, ambazo zina vikundi vya acetyl au acetoxymethyl vinavyofunika vikundi vya phenolic OH au carboxylic vya Calcein, huingia kwa uhuru ndani ya seli. Kisha esterases za ndani ya chembe huzifanya ziwe na rangi ya kijani-kibichi, ambayo huhifadhiwa na chembe. Kwa hiyo, substrates hizi za esterase zinaweza kutumika kama vipimo vya viability kwa seli. Calcium-methylamine (CMA) ni kiungo cha msingi cha esterase ya fluorescent. Kwa ajili ya kupaka rangi ya seli za viumbe hai, chombo cha kupaka rangi ya Propion iodide (PIO) na chombo cha kupaka rangi ya Calcium (CA) hutumiwa kutofautisha seli za viumbe hai na zile zilizokufa. Chembe za wafu zinaweza kugeuka rangi na iodidi ya propidium kwa sababu hupita kupitia utando wa seli ulioharibika; chembe zenye uhai zinaweza kugeuka rangi na Calcium-AM kwa sababu ya shughuli zao za esterase. Kwa kuwa wote calcine na propidium iodide-DNA complex inaweza kuwa msisimko katika 490 nm kutoa kijani na nyekundu fluorescence, kwa mtiririko huo, wafu na viable seli inaweza kuchunguzwa na microscope fluorescence. Chembe za maiti huonekana tu kwa msukumo wa 545 nm. Utafiti wa Cell Proliferation Cytotoxicity Assay Kit (CTPK) ni kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho cha kitambulisho.