Iyo Countstar system inosanganisa iyo cytometer yemufananidzo uye cell counter kuita imwe bhenji-yepamusoro chiridzwa.Iyi application-inotyairwa, compact, uye otomatiki sero imaging system inopa yese-mu-imwe mhinduro yekutsvagisa masero egomarara, kusanganisira kuverenga kwesero, kugona (AO/PI, trypan blue), apoptosis (Annexin V-FITC/PI), sero. kutenderera (PI), uye GFP/RFP kutapurirana.
Abstract
Gomarara ndicho chimwe chezvikonzero zvikuru zverufu pasi rose, uye kuvandudzwa kwenzira itsva dzekurapa gomarara kwakakosha zvikuru.Sero reCancer ndicho chinhu chekutanga chekutsvagisa chegomarara, ruzivo rwakasiyana runofanira kuongororwa kubva musero regomarara.Iyi nzvimbo yekutsvagisa inoda nekukurumidza, yakavimbika, yakapfava uye yakadzama yesero kuongororwa.Countstar system inopa yakapusa mhinduro chikuva chekuongorora cancer maseru.
Kudzidza Cancer Cell Apoptosis na Countstar Rigel
Apoptosis assays anowanzo shandiswa mumarabhoritari mazhinji nekuda kwezvinangwa zvakasiyana kubva pakuongorora hutano hwesero tsika kusvika pakuongorora kuchetura kwepanera remakomponi.
Apoptosis assay imhando inoshandiswa kuona iyo apoptosis muzana yemasero neAnnexin V-FITC/PI staining nzira.Annexin V inosungira kuphosphatidylserine (PS) nekutanga apoptosis sero kana necrosis cell.PI inongopinda necrotic/yakanyanya kunonoka-danho apoptotic masero.(Mufananidzo 1)
A: Early apoptosis Annexin V (+), PI (-)
B:Late apoptosis Annexin V (+), PI (+)
Mufananidzo1: Yakawedzerwa ruzivo rwe Countstar Rigel mifananidzo (5 x magnification) ye293 masero, akabatwa neAnnexin V FITC uye PI.
Cell Cycle Analysis yeCancer Cell
Kutenderera kwesero kana sero-kupatsanurwa kutenderera ndiko kutevedzana kwezviitiko zvinoitika musero zvinotungamira kukupatsanurwa uye kudzokororwa kweDNA yayo (DNA replication) kuburitsa vanasikana vaviri masero.Mumasero ane nucleus, semu eukaryotes, kutenderera kwesero kunokamurwawo kuita nguva nhatu: interphase, mitotic (M) phase, uye cytokinesis.Propidium iodide (PI) idhayi yenyukireya inosvibisa iyo inowanzoshandiswa kuyera kutenderera kwesero.Nekuti dhayi haigone kupinda mumasero mhenyu, maseru anogadziriswa ne ethanol isati yasvibiswa.Masero ese anobva asvibiswa.Masero anogadzirira kupatsanura achange aine huwandu hunowedzera hweDNA uye anoratidza zvakaenzana kuwedzera fluorescence.Misiyano yefluorescence intensity inoshandiswa kuona huwandu hwemasero muchikamu chimwe nechimwe chesero kutenderera.Countstar inogona kutora mufananidzo uye mhedzisiro icharatidzwa muFCS Express software.(Mufananidzo 2)
Mufananidzo 2: MCF-7 (A) uye 293T (B) yakasvibiswa nesero cycle Detection Kit ine PI, zvigumisiro zvakagadziriswa na Countstar Rigel, uye yakaongororwa neFCS express.
Viability uye GFP Transfection Determination muSero
Munguva yebioprocess, GFP inowanzoshandiswa kusanganisa neprotein recombinant sechiratidzo.Sarudza iyo GFP fluorescent inogona kuratidza chinangwa cheprotein kutaura.Countstar Rigel inopa inokurumidza uye yakapusa bvunzo yekuyedza kutapurirana kweGFP pamwe nekuita.Masero akasvibiswa nePropidium iodide (PI) uye Hoechst 33342 kutsanangura huwandu hwesero akafa uye huwandu hwesero.Countstar Rigel inopa inokurumidza, yehuwandu nzira yekuongorora GFP kutaura kunyatsoita uye kugona panguva imwe chete.(Mufananidzo 4)
Mufananidzo 4: Masero anowanikwa achishandisa Hoechst 33342 (bhuruu) uye chikamu cheGFP chinoratidza masero (girinhi) chinogona kutsanangurwa zviri nyore.Nonviable cell yakasvibiswa nepropidium iodide (PI; tsvuku).
Viability uye Cell Count
AO/PI Dual-fluoresces kuverenga imhando yeassay inoshandiswa kuona kusungwa kwesero, kugona.Iyo yakakamurwa kuita mutsara wekuverenga mutsara uye yekutanga maseru kuverenga zvichienderana neakasiyana masero.Mhinduro yacho ine musanganiswa wegreen-fluorescent nucleic acid stain, acridine orange, uye redfluorescent nucleic acid stain, propidium iodide.Propidium iodide idhayi yekusarudzika membrane inongopinda mumasero ane membrane yakakanganiswa nepo acridine orenji inopinda mumasero ese muhuwandu.Kana madhayi ese ari maviri ari munucleus, propidium iodide inokonzera kuderedzwa kweacridine orange fluorescence nefluorescence resonance energy transfer (FRET).Nekuda kweizvozvo, maseru ane nucleated ane intact membranes anoshatisa fluorescent green uye anoverengerwa semhenyu, nepo nucleated maseru ane mamembres akakanganiswa anongosvibisa fluorescent tsvuku uye anoverengwa seakafa kana uchishandisa Countstar Rigel system.Zvisiri-nucleated zvinhu zvakaita semasero matsvuku eropa, maplatelet nemarara haaite fluoresce uye anofuratirwa neCountstar Rigel software.(Mufananidzo 5)
Mufananidzo 5: Countstar yakagadziridza nzira mbiri-fluorescence yekusvibisa nzira iri nyore, chaiyo yekutsunga kwePBMC kusungwa uye kushanda.Samples dzakasvibiswa neAO/PI dzinogona kuongororwa neCounstar Rigel
