GENEWORLD QUY TRNH NH GI S TNG TRNG CA T BO (CELL GROWTH DETERMINATION)M t: Phng php MTT nh gi t bo l phng php c s dng tt nht khi nui cy t bo trn a nhiu ging. c kt qu chnh xc, s lng t bo nn c xc nh trong sut pha tng trng. Mi test nn bao gm c ging blank cha mi trng hon chnh m khng cha t bo. Ch : vic nhim cc vi khun, mycoplasma v cc vi sinh vt khc c th ct vng MTT tetrazolium; do , cc nui cy b nhim khng nn s dng phng php ny kim tra.

1. Quy trnh

1. 2. 3. 4. a.

b.

c. 5. 6. 7. 8.

Ly sn phm nui cy khi t nui, chuyn vo khu vc nui cy sch B sung MTT SOLUTION v trng mt lng bng 10% th tch cui. Chuyn sn phm nui cy vo t nui v trong 3-4 gi. Sau giai on , ly sn phm nui ra khi t nui v ha tan cc tinh th MTT formazal theo ch dn sau: i vi t bo bm dnh: loi b v x l dung dch nui. Thm MTT SOLVENT mt th tch bng vi th tch nui cy ban u. Th tch SOLVENT nn ph hp so snh d dng i vi t bo nui cy huyn ph: Trng hp ny cc tinh th MTT formazan c th mt khi loi b dung dch nui, thm MTT SOLVENT trc tip vo a nui vi lng th tch bng th tch nui cy ban u. Kt qu nn c phn tch trong vng 1 gi sau khi thm MTT SOLVENT. Khuy nh trong mt h thng lc lun chuyn tng cng kh nng ha tan. Thnh thong c th s dng pipette trn u cc tinh th MTT formazan ha tan hon ton, c bit khi nui cy t bo mt cao. o hp thu quang ph bc sng 570 nm (bc sng kim tra). o hp th nn 690 nm (bc sng tham chiu). Cc th nghim c thc hin trong a 96 ging cng c th c o bng mt u c a ELISA c trang b vi cc b lc thch hp. Vi cc xt nghim thc hin trong cc a a ging khc s yu cu chuyn sang cc cuvet hoc a vi kch thc thch hp cho c quang ph. CH : vic chuyn i MTT thnh MTT formazan thay i theo tng loi t bo. Do tnh a dng m c th c s khc nhau v trng thi sinh l ca cc chng t bo thuc cng mt dng t bo, do gi tr OD/s lng t bo s khc nhau i vi mi kiu b tr th nghim khc nhau.

2. Cc nguyn nhn c th gy sai s

1. MTT SOLVENT n nh khi lu tr ng lnh. Bo qun 2-8 C c th dn n phn hy v mang li kt qu sai lch. Sn phm chuyn sang mu ti hoc hnh thnh cc tinh th l biu hin cho thy sn phm b h hng. 2. S nhim vi sinh vt s lm phn ct MTT v hnh thnh nn MTT formazan dn n nhng sai s trong kt qu. 3. S bay hi khng ng u ca dung dch nui cy trong mi ging ca a a ging c th gy ra kt qu sai lch. 4. Mc protein cao (huyt thanh, albumin...) trong mi trng nui cy c th to kt ta khi MTT SOLVENT c thm vo. Mu vi nng protein tng ng 10% huyt thanh thai b c th chp nhn c. Sera vi nng protein cao hn so vi huyt thanh thai b c th c s dng vi t l phn trm thp hn. 1. 2. 3. 4. Slater, T. et al. (1963) Biochem. Biophys. Acta 77:383. Mossman, T. (1983) J. Immunol. Methods 65:55. Denizot, F. and Lang, R. (1986) J. Immunol. Methods 89:271. Carmichael, J. et al. (1987) Cancer Research 47:936

3. Than kho

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