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need help on Macrophage RAW 264.7 cells

need help on Macrophage RAW 264.7 cells# Biology - 生物学

c*l2011-07-01 07:07

1 楼

3月开卡，第一个账单刚出来
5%的category都没问题，但是我有一笔在walmart的交易，$4.52
应该是算在all other 1% cashback里面的吧，可是cashback detail里对应的项却只有
$0.01的cashback，不应该是$0.04吗？
不是纠结这点cashback，只是想弄清楚它是怎么算的
多谢！

S*r2011-07-01 07:07

2 楼

We performed a reporter assay on RAW cells days ago. The transfection
efficiency is fine, around 10-15% with EGFP. However, luciferase value is
extremely low, including both Firefly and Renilla that acts as the internal
control. I wonder if an activation is required for these cells, just like
handling in T cells that requires PMA and Ionomycin?
If it requires, what's concentration should I use?
Thanks in advance!

c*l2011-07-01 07:07

3 楼

靠,发现了... term里写的是“up to 1%”...

【在 c******l 的大作中提到】

: 3月开卡，第一个账单刚出来
: 5%的category都没问题，但是我有一笔在walmart的交易，$4.52
: 应该是算在all other 1% cashback里面的吧，可是cashback detail里对应的项却只有
: $0.01的cashback，不应该是$0.04吗？
: 不是纠结这点cashback，只是想弄清楚它是怎么算的
: 多谢！

a*g2011-07-01 07:07

4 楼

10-15% is not a fine transfection efficiency!!!
However, for RAW cells, it is normal. As a result, low luc value is not
unexpected. You can do nothing about it without increasing your transfection
efficiency at the first place.

internal

【在 S****r 的大作中提到】

: We performed a reporter assay on RAW cells days ago. The transfection
: efficiency is fine, around 10-15% with EGFP. However, luciferase value is
: extremely low, including both Firefly and Renilla that acts as the internal
: control. I wonder if an activation is required for these cells, just like
: handling in T cells that requires PMA and Ionomycin?
: If it requires, what's concentration should I use?
: Thanks in advance!
:

u*n2011-07-01 07:07

5 楼

walmart已经很便宜了，估计没啥可以返的了，还不如买walmart的giftcard

S*r2011-07-01 07:07

6 楼

Thanks! We tried in two other cell lines, both with similar transfection
efficiency, but still can see Renilla value from 200 to 1000, while in RAW
cells, it's only around 10.
I'm also not sure if RAW is tougher to be lysed in passive lysis buffer.
Should I try freezing-thawing cycle to disrupt them?

transfection

【在 a******g 的大作中提到】

: 10-15% is not a fine transfection efficiency!!!
: However, for RAW cells, it is normal. As a result, low luc value is not
: unexpected. You can do nothing about it without increasing your transfection
: efficiency at the first place.
:
: internal

L*n2011-07-01 07:07

7 楼

discover教会了很多人看条款要仔细……

【在 c******l 的大作中提到】

: 靠,发现了... term里写的是“up to 1%”...

a*g2011-07-01 07:07

8 楼

That definitely helps. However, given such a low transfection efficiency, I
personally don't trust those luc data although many paper showed nice
results from this assay.

【在 S****r 的大作中提到】

: Thanks! We tried in two other cell lines, both with similar transfection
: efficiency, but still can see Renilla value from 200 to 1000, while in RAW
: cells, it's only around 10.
: I'm also not sure if RAW is tougher to be lysed in passive lysis buffer.
: Should I try freezing-thawing cycle to disrupt them?
:
: transfection

c*l2011-07-01 07:07

9 楼

唉。。。这个up to真是太恶心了。。。

【在 L*******n 的大作中提到】

: discover教会了很多人看条款要仔细……

D*g2011-07-01 07:07

10 楼

You can try electropotration to increase the transfection efficiency, such
as the neon system from Invitrogen or Nucleofector from Amaxa, you can
easily transfect the cells for more than 40-50%.

internal

【在 S****r 的大作中提到】