counting viable cells directly from wells - (Aug/01/2007 )
Has anyone try to count viable cells directly (using trypan blue) from the well without using hemacytometer? What is the protocol involved and how to determine the cell viability as it is harder to count from the well compare to hemacytometer?
need help urgently. thank you.
-sanjiun81-
if cells are in monolayer and if trypan blue does not detach them it will be ok to count them directly from wells.
but you'll need something for limiting the area of counting in a precise manner.
-fred_33-
QUOTE (fred_33 @ Aug 1 2007, 01:24 AM)
if cells are in monolayer and if trypan blue does not detach them it will be ok to count them directly from wells.
but you'll need something for limiting the area of counting in a precise manner.
but you'll need something for limiting the area of counting in a precise manner.
What Fred 33 is talking about is a GRATICULE. It is a device which will grid your monolayer into a known area. What you must not do is introduce BIAS into your experiment i.e. if you set up the experiement YOU HAVE TO GET SOMEONE ELSE TO COUNT YOUR CELLS. Also you have to count at least 3 different areas if you want to do Statistics on them later.
-Rhombus-
rhank you rhombus for the precision.
-fred_33-
QUOTE (sanjiun81 @ Aug 1 2007, 01:42 AM)
Has anyone try to count viable cells directly (using trypan blue) from the well without using hemacytometer? What is the protocol involved and how to determine the cell viability as it is harder to count from the well compare to hemacytometer?
need help urgently. thank you.
need help urgently. thank you.
For some cell lines, we would take a picture of the well at 40x. Now the image is stored and you can count your cells one by one at your own pace.
-scolix-
QUOTE (Rhombus @ Aug 1 2007, 01:30 AM)
What Fred 33 is talking about is a GRATICULE. It is a device which will grid your monolayer into a known area. What you must not do is introduce BIAS into your experiment i.e. if you set up the experiement YOU HAVE TO GET SOMEONE ELSE TO COUNT YOUR CELLS. Also you have to count at least 3 different areas if you want to do Statistics on them later.
sometimes when we seed cells in well, the region at the edge will get more cells (uneven seeding of cells)... so how do you determine where you want to count? eg. you count 3 areas... all at the middle or including middle and also edge?
-sanjiun81-
i would choose same repartition for all experiments.
of course edge and center. the third region?...well doesn't truly matters, just keep it same for each wells.
-fred_33-
QUOTE (fred_33 @ Aug 2 2007, 01:03 AM)
i would choose same repartition for all experiments.
of course edge and center. the third region?...well doesn't truly matters, just keep it same for each wells.
of course edge and center. the third region?...well doesn't truly matters, just keep it same for each wells.
Dear All,
Using a normal eyepiece Graticule, with a 6 well plate and under x20 lense, the number of fields is immense. What we have done in the past is to RANDOMLY move the plate in order not to introduce bias. Also the most important thing is to have someone else do the counting. If you have set up the plate and know what you are expecting then you are introducing bias into your experiment. Remember that 3 fields is the MINIMUM, we normally look at between 5 and 10 fields/well....each of course done in triplicate.
-Rhombus-