Izeluleko eziyisisekelo zokulondolozwa kwama-cropresses

Izeluleko eziyisisekelo zokulondolozwa kwama-cropresses

Amaseli aqhubeka nemetabolism yawo ngesikhathi semisebenzi evamile yokukhula, edinga ukubamba iqhaza kwamaphrofethe ahlukahlukene. Lapho amazinga okushisa ehla ngezansi -70 ° C, ama-proteries ayeke ukusebenza. Ngakho-ke, endaweni yokushisa ephansi kakhulu, amaseli angamisa imisebenzi yawo metabolic bese ufaka isimo esishisayo esivumela ukugcinwa isikhathi eside.

1. Izinto zokuhlola

Isisekelo sezinto:

Isiko eliyisisekelo eliphakathi nendawo

I-Serum (ama-FBS ajwayelekile / NBS)

I-Ultrapure Workbench

Oyinyumba dimethyl sulfoxide (DMSO)

I-Phosbele Pbs Phosphate Buffer Solution Solution

Isibhamu sePipetting

Isibhamu sePipetting Electric

Ukulungiselela izivivinyo

a) Ukulungiselela ikhambi leqhwa:

Amaseli ajwayelekile: 55% Baseal Medium + 40% Bovine Serum (FBS / NBS) + 5% DMSO

Amaseli Abalulekile: 90% Bovine Serum (FBS / NBS) + 10% DMSO

I-Aliquot Isixazululo sokulondolozwa kwe-CRICEDED kwi-15 ml centrifuge tube [isidleke] bese sigcina ku-4 ° C ukuze isetshenziswe kamuva.

(b) Ukulungiselela amaseli okuba yiqhwa:

Ngaphambi kokuqaqa amaseli, khetha amaseli ngesimo sokukhula okuhle nakusigaba sokukhula kwe-logarithmic bese ubeka esikhundleni samahora amasha aphakathi kwamamitha ayi-12 ngaphambi kokugcina isimo se-cell.

2. Inqubo yokuhlola

1. Bheka ubuntu bamaseli ukuze ube yiqhwa, elingaba ngu-80% ~ 90%. Sebenzisa isibhamu se-pipette ukufisa okuphakathi nendawo, engeza ama-pbs oyinyumba ukuze ugeze amaseli izikhathi 1-2, bese ususa okuphakathi nendawo esimweni sesiko.

2. Faka inani elifanele le-trypsin noma ijusi lokugaya ukuvumela i-trypsin ukuthi ingene kumaseli, bese uyibeka ku-incubator yokugayeka. Bheka isimo samaseli ngaphansi kwesibonakhulu: i-cytoplasm iyabuyisa futhi amaseli awasaxhunyiwe kumashidi. Ngalesi sikhathi, engeza ikhambi lokumisa ukumisa inqubo yokugaya.

3. Bubble ngobumnene amaseli anethiphu ukwenza ukumiswa kwamaseli kanye ne-centrifuge ukumiswa kweseli ku-1000 RPM imizuzu engu-1000.

Lahla isikhulu, engeza inani elifanele lesixazululo seqhwa, futhi u-pipete ngobumnene ukwenza amaseli abala ngokulinganayo. Lungisa inqwaba yeseli nge-Cropressoalket Medium ukwenza ubuningi bokugcina 5 × 106 / ml ~ 1 × 107 / ml.

4. Sebenzisa isibhamu se-pipette ukuhlukanisa amashubhu ama-cryogenic ngokuya ngenani elilindelekile, futhi usebenzise umshini wokukhokhisa othomathikhi ozosebenza ngokuzenzakalela u-cap kanye nophawu.

5. Uhlelo lokulondolozwa okujwayelekile luyinani elipholile le -1 ° C kuya ku-2 ° C / min, futhi amaseli okuba yi-Frozen angaqashwa kancane kancane ngokwezinyathelo ezilandelayo: Ukushisa kwegumbi → 4min) → 20 ° C (30min) → -80 ° C ° C (Overnight) → Isitoreji eside ku-nitrogen ewuketshezi.

Ingagcinwa ngqo efrijini e-Freezer at -80 ° C ngobusuku bese idluliselwa ku-nitrogen ewuketshezi ukuze isitoreji.

I-Yongyue Medical iyibhizinisi eliphakeme lobuchwepheshe obukhethekile ku-R & D, ukukhiqizwa kanye nokuthengisa kokudla kwe-biomedical. Amathiphu wePipette, ama-Pipette, ipuleti le-PCR, imikhiqizo yesiko leseli nezinye izinto zokudla zebhanoyi. I-Yongyue Medical ifanelwe ukwethembana nokukhetha kwakho! Uyemukelwa!