d****i 发帖数: 2346 | 1 比如18链,16链,用来treat细胞。
求详细一点的protocol。
谢谢! | h******y 发帖数: 351 | 2 What kind of cells, which fatty acids are you using? Please give more
details.
Here is what I used for hepatocyte.
Preparation of the fatty acid stocks
The free fatty acids are prepared by dissolving each individual free fatty
acid in 100% ethanol. Most are readily dissolved in ethanol, though some
require heating to achieve fully solubility.
A final concentration of 100 mM for each of the fatty acids as follows:
31 ul 1 M Palmitic Acid Solid, 1 g / 3.3 ml EtOH
(Heat to 50C in water bath to dissolve. )
2.8 ul 1 M Palmitoleic Acid Liquid, 1 ml(1g)+2.93ml EtOH
76.9 ul 151 mM Stearic Acid Solid, 168 mg / 3.9 ml EtOH
13.4 ul 1 M Oleic Acid Liquid, 1 ml(1g)+2.54ml EtOH
35.6 ul 1M linoleic Acid Liquid, 1 ml(g)+ 2.6 ml EtOH
5.6 ul 1M Linolenic Acid Liquid, 1 ml(1g) + 2.59 ml EtOH
834.7 ul 100% EtOH
Reference:
Chessebeuf, M., and Padieu, P. (1984). Rat liver epithelial cell cultures in
a serum-free medium: Primary cultures and derived cell lines expressing
differentiated functions. In Vitro 20, 780–795.
【在 d****i 的大作中提到】 : 比如18链,16链,用来treat细胞。 : 求详细一点的protocol。 : 谢谢!
| d****i 发帖数: 2346 | 3 非常感谢!
我们用的是palmitic Acid和Stearic Acid两种,分别去treatmacrophage。据说是先用
乙醇溶解,然后再加入BSA,但是我不太明白你这个protocol。
1. 前面那个31uL、2.8uL。。。是什么的体积?
2. 2.8 ul 1 M Palmitoleic Acid Liquid, 1 ml(1g)+2.93ml EtOH
这里面 1 M Palmitoleic Acid是配好后的浓度? 1 ml(1g)+2.93ml EtOH里面的1 ml(
1g)的1mL和1g分别是啥意思?
再次感谢!
follows:
【在 h******y 的大作中提到】 : What kind of cells, which fatty acids are you using? Please give more : details. : Here is what I used for hepatocyte. : Preparation of the fatty acid stocks : The free fatty acids are prepared by dissolving each individual free fatty : acid in 100% ethanol. Most are readily dissolved in ethanol, though some : require heating to achieve fully solubility. : A final concentration of 100 mM for each of the fatty acids as follows: : 31 ul 1 M Palmitic Acid Solid, 1 g / 3.3 ml EtOH : (Heat to 50C in water bath to dissolve. )
| h******y 发帖数: 351 | 4 这几种脂肪酸中,Palmitic Acid 和 Stearic Acid常温下是固体,计算摩尔浓度比较
容易。其他几种常温下是液体,但是大体上他们的密度和水的密度很相近,1ml近似1g
。这些脂肪酸都极易溶于乙醇。溶解Palmitic Acid需要略微加热(50C)。
The free fatty acids are toxic unless they are presented bound to purified,
fatty acid free, endotoxin-free serum albumin, which is used at a typical
concentration of 0.1–0.2%.
Take 31ul 1M palmitic acid, 2.8ul 1M Palmitoleic acid etc and bring the
final volume to 1000ul with 100% EtOH, you will get 0.1M FFA. Add 76ul of
the mixture to 1L culture medium and you will get 7.6uEq/l, which is good
for hepatocyte culture. However, your particular macrophage culture
condition might be different and you are encouraged to refer to publications
in your own field.
【在 d****i 的大作中提到】 : 非常感谢! : 我们用的是palmitic Acid和Stearic Acid两种,分别去treatmacrophage。据说是先用 : 乙醇溶解,然后再加入BSA,但是我不太明白你这个protocol。 : 1. 前面那个31uL、2.8uL。。。是什么的体积? : 2. 2.8 ul 1 M Palmitoleic Acid Liquid, 1 ml(1g)+2.93ml EtOH : 这里面 1 M Palmitoleic Acid是配好后的浓度? 1 ml(1g)+2.93ml EtOH里面的1 ml( : 1g)的1mL和1g分别是啥意思? : 再次感谢! : : follows:
| d****i 发帖数: 2346 | 5 谢谢!我再去找相关文献看看。
这个对照应该用0.1–0.2%的BSA吧? | h******y 发帖数: 351 | 6 I think so. Remember to use fatty acid-free BSA (Sigma, A8806) since you are
looking at the effect of exogenous fatty acid.
【在 d****i 的大作中提到】 : 谢谢!我再去找相关文献看看。 : 这个对照应该用0.1–0.2%的BSA吧?
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