Product Format: a T25 flask
Organism: Homo sapiens (human)
Complete Growth Medium: See Propagation
Source: Organ: breast Tissue: mammary Disease: normal Cell Type: epithelial
Atmosphere: air, 95%; carbon dioxide (CO2), 5% ，37℃
Application: Cells and cancer research
Propagation: The base medium for HMEC is formulated RPMI-1640 Medium. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to
a final concentration of 10%.
NOTE: FOR RESEARCH USE ONLY.
Components ：Item a T25 flask Manual
Specifications： 2X106 1copy
Subculturing: Protocol:
1.Remove culture medium to a centrifuge tube.
2.Briefly rinse the Cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3.Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until Cell layer is dispersed (usually within 1 to 5 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37C to facilitate dispersal.
4.Add 6.0 to 8.0ml of complete growth medium and aspirate cells by gently pipetting.
5.Transfer the Cell suspension to the centrifuge tube with the medium and cells from
step 1, and centrifuge at approximately 125 x g for 5 to 10 minutes. Discard the supernatant.
6.Resuspend the Cell pellet in fresh growth medium. Add appropriate aliquots of the Cell suspension to new culture vessels.
7.Incubate cultures at 37C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:5 is recommended
Medium Renewal: Every 2 to 3 days
Preservation: Freeze medium: Culture medium,90%; DMSO, 10%
Storage temperature: liquid nitrogen vapor phase
Tips：
1. 細(xì)胞經(jīng)過運(yùn)輸后，部分細(xì)胞由于溫度變化及劇烈碰撞死亡破碎形成碎片，是正?，F(xiàn)象。貼壁細(xì)胞可以消化，懸浮細(xì)胞直接混勻收集細(xì)胞，900-1000rpm(約150g)離心3min，棄上清。加5ml PBS重懸細(xì)胞，再900-1000rpm(約150g)離心3min，用新鮮的完全培養(yǎng)基重懸接種到新的培養(yǎng)瓶。第二次PBS重懸是為了去除碎片，如果平時(shí)碎片比較少，傳代時(shí)可以省略PBS重懸的步驟；如果碎片很多，建議PBS多洗幾次。
2. 細(xì)胞生長不均時(shí)，可以將細(xì)胞消化吹散后加入新的培養(yǎng)基重新接種或傳代。
3. 細(xì)胞生長緩慢時(shí)，可以選擇提高血清濃度培養(yǎng)（最高不超過20%），也可以根據(jù)細(xì)胞生長狀態(tài)，選擇傳代細(xì)胞到新的培養(yǎng)瓶中繼續(xù)培養(yǎng)。
4. 不同細(xì)胞貼壁性差異比較大，所以消化時(shí)間差別較大，20s-10min均有可能，具體以細(xì)胞消化到相互分離但未脫落，并可以輕輕吹下為準(zhǔn)，嚴(yán)禁消化到細(xì)胞完全漂浮。客戶消化過度導(dǎo)致細(xì)胞死亡、漂浮、生長緩慢，不提供免費(fèi)售后服務(wù)。
5. 干冰發(fā)貨均為兩支，客戶先復(fù)蘇一支，若復(fù)蘇失敗及時(shí)聯(lián)系我方并在我方指導(dǎo)下復(fù)蘇第二支。
6. 細(xì)胞狀態(tài)正常時(shí)，應(yīng)盡快凍存細(xì)胞保種，凍存后應(yīng)隨機(jī)抽取一支檢測凍存效果。
