y****6
发帖数: 196 | 1 HEK293 cells in 96-well plate, DMEM with 10% FBS, cultured 2d, about 100%
confluency.
Washed once with PBS before adding fixation solution (3.7% formaldehyde/PBS).
Cells detached in fixastion solution. Is it because formaldehyde does not
work with HEK293 cells? Anyone tried methenol? What's the concentration? |
h******y
发帖数: 351 | 2 Do not wash your cells with PBS as 293 cells are easy to detach. Add
fixative after remove medium, or pre-coat your plate with poly-D/L-Lysine.
PBS).
【在 y****6 的大作中提到】
: HEK293 cells in 96-well plate, DMEM with 10% FBS, cultured 2d, about 100%
: confluency.
: Washed once with PBS before adding fixation solution (3.7% formaldehyde/PBS).
: Cells detached in fixastion solution. Is it because formaldehyde does not
: work with HEK293 cells? Anyone tried methenol? What's the concentration?
|
V********n
发帖数: 305 | |
y****6
发帖数: 196 | 4 Can anyone explain how fixation works? My understanding is that fixative
helps the crosslink of proteins and other macromolecules. I also have an
impression that it helps the cells to attach to the plate more firmly. It is
correct? |
c**n
发帖数: 73 | 5 I use 4% Paraformaldehyde to fix 293 HEK cells for 15 min.
I always put cells on cover slips coated with fibronectin or polyL lysin and
then fix them. It works fine.
PBS).
【在 y****6 的大作中提到】
: HEK293 cells in 96-well plate, DMEM with 10% FBS, cultured 2d, about 100%
: confluency.
: Washed once with PBS before adding fixation solution (3.7% formaldehyde/PBS).
: Cells detached in fixastion solution. Is it because formaldehyde does not
: work with HEK293 cells? Anyone tried methenol? What's the concentration?
|
h****n
发帖数: 2552 | 6 Fixation reagent is no use to help cells attch on a plate. I suggest coating
your
plate with poly-lysine
is
【在 y****6 的大作中提到】
: Can anyone explain how fixation works? My understanding is that fixative
: helps the crosslink of proteins and other macromolecules. I also have an
: impression that it helps the cells to attach to the plate more firmly. It is
: correct?
|
b*****l
发帖数: 9499 | 7 不要用 PBS，因为里面没 Ca/Mg，会导致细胞 detach。
用 DPBS with Ca/Mg 来洗细胞，然后再 fix。
而且 PBS 太 hush。原则是活细胞用 DPBS，死细胞或者不关细胞才用 PBS。
PBS).
【在 y****6 的大作中提到】
: HEK293 cells in 96-well plate, DMEM with 10% FBS, cultured 2d, about 100%
: confluency.
: Washed once with PBS before adding fixation solution (3.7% formaldehyde/PBS).
: Cells detached in fixastion solution. Is it because formaldehyde does not
: work with HEK293 cells? Anyone tried methenol? What's the concentration?
|
y****6
发帖数: 196 | 8 Thanks a lot for the reply from all of you. I will try coating. |
