Peripheral blood mononuclear cells (PBMCs) inowanzogadziriswa kuti iparadzanise kubva kuropa rose ne density gradient centrifugation.Masero iwayo anosanganisira lymphocytes (T masero, B masero, NK masero) uye monocytes, inowanzoshandiswa mumunda immunology, sero kurapa, chirwere chinotapukira uye vaccine kukura.Kuongorora uye kuongorora kugona uye kusungirirwa kwePBMC kwakakosha kumakiriniki marabhoritari, yekutanga sainzi yesainzi yekutsvagisa uye immune cell kugadzirwa.
Fig 1. Isolated PBMC kubva muropa idzva rine Density gradient centrifugation
AOPI Dual-fluoresces kuverenga imhando yeassay inoshandiswa kuona kusungwa kwesero uye kushanda.Mhinduro isanganiswa yeacridine orange (iyo green-fluorescent nucleic acid stain) uye propidium iodide (iyo red-fluorescent nucleic acid stain).Propidium iodide (PI) idhayi yekusarudzika membrane inongopinda mumasero ane membrane yakakanganiswa, nepo acridine orenji inokwanisa kupinda mumasero ese muhuwandu.Kana madhayi ese ari maviri ari munucleus, propidium iodide inokonzera kuderedzwa kweacridine orange fluorescence nefluorescence resonance energy transfer (FRET).Nekuda kweizvozvo, masero ane nucleated ane intact membranes anoshatisa fluorescent green uye anoverengerwa semhenyu, nepo nucleated maseru ane membrane akakanganiswa anongosvibisa fluorescent tsvuku uye anoverengwa seakafa kana uchishandisa Countstar® FL system.Zvinhu zvisina nucleated zvakaita semasero matsvuku eropa, maplatelet nemarara haaite fluoresce uye anofuratirwa neCountstar® FL software.
Maitiro ekuedza:
1.Dilute sampu yePBMC muzvikamu zvishanu zvakasiyana nePBS;
2.Wedzera 12µl AO/PI mhinduro mu12µl sampuli, zvinyoronyoro yakasanganiswa nepipette;
3.Dhirora 20µl musanganiswa mukamuri siraidhi;
4.Bvumira masero kuti agare mukamuri kweinenge 1 miniti;
5.Insect the slide kupinda Countstar FL chiridzwa;
6.Sarudza "AO / PI Viability" assay, wozoedza ne Countstar FL.
Yambiro: AO uye PI inogona kukonzera kenza.Zvinokurudzirwa kuti anoshanda apfeke midziyo yekudzivirira (PPE) kudzivirira zvakanangana neganda nemeso.
Mhedzisiro:
1.Bright Field uye Fluorescence mifananidzo yePBMC
Iyo AO nePI dhayi zvese zvinosvibisa DNA musero nucleus yemasero.Naizvozvo, maPlatelets, masero matsvuku eropa, kana marara emasero haakwanise kukanganisa kutariswa kwePBMC uye mhedzisiro.Maseru mapenyu, masero akafa uye marara anogona kuve nyore kusiyaniswa hwaro pamifananidzo inogadzirwa neCountstar FL (Mufananidzo 1).
Mufananidzo 2.Bright Field uye Fluorescence mifananidzo yePBMC
2.Concentration uye Viability yePBMC
Iwo masampula ePBMC akaderedzwa muna 2, 4, 8 uye 16 nguva nePBS, ipapo iwo masampuli akaiswa musanganiswa weAO/PI wedhayi uye akaongororwa naCountstar FL zvichiteerana.Mhedzisiro yekuisa pfungwa uye kugona kwePBMC inoratidzwa senge pazasi nhamba:
