讨论：Hela细胞不同条件下的成球状况

ziyunfei1982

CD133就是个衡量指标而已，致瘤性往裸鼠体内打多了一般得HeLa都能长成瘤子，CD133分选的话，可能是CD133阳性细胞群恶性度更高吧。单纯的想弄悬浮的细胞球，换HeLa细胞系的一个亚型HeLa S3，用专用的悬浮培养基，加大浓度的生长因子，成球没问题。
2 N! K9 o9 t$ S( \( ^但是你要想说CD133是肿瘤干细胞的一个标记的话，甚至是HeLa的话，恐怕没什么新意

drgaoh

分选好做吗，用的什么方法，试剂贵不贵

yxc

     在后两张图里面，我并没有看到典型的细胞球，的确有些细胞聚集在一起，但是形态很不规则，没有很圆的球体，猜猜是细胞密度太大或者没有混匀的结果，建议重新传一次看看。

yxc

回复 drgaoh 的帖子8 H1 d1 Q/ S# Z: e1 ]7 T3 ^

* n( W' S" g' w7 @, @4 w. e1. magnetic bead cell sorting- g; K5 O' A8 j" n8 @; h

% S. x  m. J+ T1 pMagnetic-activated cell sorting (MACS) is a trademark name for a method for separation of various cell populations depending on their surface antigens (CD molecules). The term MACS is a registered trademark of Miltenyi Biotec.
2 T7 H0 l5 W( t. c& jMagnetic cell separation using antibodies is also possible using monosized magnetic beads (Dynabeads) from Invitrogen (now part of Life Technologies). The Dynabeads technology is not column based - instead these magnetic beads with attached cells enjoy liquid phase kinetics in a sample tube, and the cells are isolated by placing the tube on a magnetic rack.
. |( K/ P- g" F$ B! I; mProcedure1 I, x- g/ c- O
The MACS method allows cells to be separated by incubating with magnetic nanoparticles coated with antibodies against a particular surface antigen. This causes the cells expressing this antigen to attach to the magnetic nanoparticles. Afterwards the cell solution is transferred on a column placed in a strong magnetic field. In this step, the cells attached to the nanoparticles(expressing the antigen) stay on the column, while other cells (not expressing the antigen) flow through. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s).
' F! a& y1 [4 v) f: FPositive selection
( k" E7 Q2 U3 o9 d1 y% i$ `' [In positive selection the cells expressing the antigen(s) of interest, which attached to the magnetic column, are washed out to a separate vessel, after removing the column from the magnetic field. This method is useful for isolation of a particular cell type, for instance CD4 lymphocytes.3 v" [1 u& y, f9 H5 [. Q2 a
Negative selection
9 V$ j, _$ M* _0 B$ X' N& KIn negative selection the antibody used is against surface antigen(s) which are known to be present on cells that are not of interest. After administration of the cells/magnetic nanoparticles solution onto the column the cells expressing these antigens bind to the column and fraction that goes through is collected, as it contains almost no cells with undesired antigens.
% ~2 _6 m* {: `: z/ aModifications' k$ o  R$ f. M: S' E5 I9 a
Magnetic nanoparticles conjugated to an antibody against an antigen of interest are not always available, but there is a way to circumvent it. Since fluorophore-conjugated antibodies are much more prevalent, it is possible to use magnetic nanoparticles coated with anti-fluorochrome antibodies. They are incubated with the fluorescent-labelled antibodies against the antigen of interest and may thus serve for cell separation with respect to the antigen.

yxc

2.Fluorescence-activated cell sorting
6 `- \5 q: u" M& }/ S  @0 tfrom：Wikipedia8 a1 p9 r' T1 L  ?- p

2 h, k6 t1 E! Q+ x( T6 a2 U; `Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The acronym FACS is trademarked and owned by Becton, Dickinson and Company.[12] While many immunologists use this term frequently for all types of sorting and non-sorting applications, it is not a generic term for flow cytometry. The first cell sorter was invented by Mack Fulwyler in 1965, using the Coulter principle, a relatively difficult technique and one no longer used in modern instruments. The technique was expanded by Len Herzenberg, who was responsible for coining the term FACS. Herzenberg won the Kyoto Prize in 2006 for his work in flow cytometry.+ m+ F3 N& J0 d- Y* f( @) Z: r
The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring is placed just at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.

wolf1978

CD133是常用的肿瘤干细胞分选标志啊！你这结果蛮在意料之中的啊！需要进行深入的细胞行为效应检测才好啊！

Tonypan

Hela表达CD133嘛？以前有检测过CD133在Caski和Hela中的表达情况，和同型对照没区别

shengxiaqq

前两年M. H. Wright等通过一些证据证明了CD133是一个亚组的乳癌干细胞，而楼主这一现象似乎正是在证实这一点。当然，其鉴定不能仅仅这样就可以的，可以通过其潜在的增殖能力，分化特性来进一步认证，而对于小鼠成瘤这个标准似乎是一般的肿瘤细胞都可以有成瘤的能力吧。

sjmpt

金标准就是干细胞小鼠成瘤实验！！！我们也这么认为

jkfly

CD133阳性是肿瘤干细胞的非充分非必要条件，建议先养成球后再流式分选，看CD133的丰度