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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