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发表于 2012-8-1 09:32:07
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gcz5340 发表于 2012-7-31 23:01
自己顶一下，这段还是讨论下：
“如果你准备用于标记1亿个细胞，千万不要将抗体用量也增加100倍 ...
原文找到了，我以前翻译的版本见：http://www.flowcyto.cn/bbs/forum.php?mod=viewthread&tid=290 Mario Roederer认为还是跟染色体积关系更大，而细胞数量相对来说影响偏小，这其实我觉得牵涉到抗体染色浓度吧。这个深有体会，就是我上面这个回复中提到，那几次忘记稀释红细胞的情况，红细胞数量相差100倍，但荧光变化不大，除了抗体效价可能高之外，应该证实了文中所讲的跟染色体积关系更大。
Antibody Titration Protocol
Dr. Mario Roederer
Note that this information is for any and all antibodies--conjugate or unconjugated. Always titrate your antibodies, even commercial antibodies. Do NOT rely on what your manufacturer's instructions state--not because they are wrong, but because the manufacturer did NOT titrate the antibody under the same conditions that you are using! Antibody titrations are NOT expressed as antibody mass per number of cells. The relevant value is antibody concentration, i.e., ug per mL. The number of cells that is stained is nearly irrelevant; you will find the same titration behavior whether you stain 100,000 or 1 million cells.
Generally, start titrations at about 10 ug per ml, and do 8 2-fold dilutions. Assuming staining in 100 ul (generally), start by putting 2 ug in 100 ul of the first well, then remove 50 ul and add it to 50 ul of diluent in the second well; take 50 of that and add to 50 of the third well, etc. down the line. Then come back and add 50 ul of cells to each well. Note two different concentrations that you might use: one is the "saturating" concentration (the lowest concentration which gives you nearly maximal fluorescence), and the other is the "separating" concentration. This latter is a subjective decision on which concentration works the "best"--gives you good separation, low background. For some antibodies, this might be much less than saturating. However, you should know that at less than the saturating concentration, your final staining intensity will be time and temperature dependent.
Titrate every reagent under the conditions you plan to use it. Titrations will be temperature, time, and condition-dependent. You can have a different titration value for fix/perm protocols than you do for simple surface staining; different titration for human vs. monkey cells.
If you plan to stain 100 million cells, do NOT INCREASE the antibody by 100-fold. Generally increase it 2-5 fold, but even 1x will often be enough. If you routinely stain 100 million cells (for a sort, for example), it might be useful to do a quick, 2- or 3-point titration on that many cells. Likewise, if you stain only 50,000 cells, do NOT DECRASE the antibody amount. Remember, the relevant antibody amount to use is
CONCENTRATION, expressed as amount of antibody per staining volume. This does mean that if you titrate in 100 ul and stain in 1 ml, you will need to use 10x the antibody. Likewise, if you titrate in 200 ul and stain in 50 ul, you can use 1/4th the antibody.
The antibody titer you choose will depend on your staining time. There is no reason you can't choose a 15 min incubation for staining; it simply requires somewhat higher antibody concentration than 30 or 60 min. In fact, you could choose a concentration that works well at 3 min if you so wish!
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发表于 2012-7-31 22:52:44
