HEMOCYTOMETER - (Oct/26/2006 )

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This was really very Helpful

Thanks:)

Pooja

QUOTE (Jou @ Oct 26 2006, 10:40 AM)

Suppose you have 5ml of your cell suspension containing 2.3 x 10^6 cells/ml
Then:

10^4 / 2.3 x 10^6 x Z ml = 5ml

Where Z is the amount of medium you have to add to obtain the desired 10^4 cells/ml.

Z = 5/(10^4/2.3 x 10^6) = 5 x 2.3 x 10^6 / 10^4 = 5 x 2.3 x 10^2 = 1150 ml.

So it is a 1:230 ratio (1150/5). Accordingly you can add 1µl of your initial suspension (2.3 x 10^6) to 229 µl of medium and obtain 230 µl which contain 10^4 cells.

-p.maj-

Thanks for the information:)

I had a question, Ideally how many cells should be plated for assays such as ELISA using a 96-well plate?

Pooja

QUOTE (Rhombus @ Oct 26 2006, 11:09 AM)

Dear BioDude 1,

First of all 230 cells/ square (16 smaller squares) is to many cells to do an accurate cell count. You should really have between 25 and 100 cells/square. And you should count 4 squares and then average the count.
Are you doing a VIABLE count i.e. using Trypan blue as an EXCLUSION DYE. If you are you need to have the dilution factor within your calculation ......for example 100ul of cell suspension + 100ul of Trypan is a 1:2 dilution, thus the factor of 2 is put into the following calculation :

Mean Cells/square x 2 x 10,000 (10^4) = No of cells/ ml.

Your example:

230 x 10,000 = 2.3 x 10^6

You need to dilute your cell suspension by a factor of 230 to get a cell suspension of 10^4

i.e 1ml of cell suspension to 229mls of media = 10^4 cells/ml

or 0.1ml of cell suspension to 22.9mls of media = 10^4 cells/ml

or 10ul of cell suspension to 2.29mls of media = 10^4 cells/ml

or as Jou has already stated eloquently 1ul of cell suspension to 229ul of media = 10^4 cells

Hope this is clear

Rhombus.

-p.maj-

Hi , I have a question regarding cell counting and the dye exclusion test :

I have always used the dilution factor when doing the counting , but the lab I am in right now the calculation that they do am not able to follow –
I ‘ve been told to count the number of cells in any of the 2 opposite squares take their average .Take the average cell count and multiply it with 10 ^ 4

i.e. if I get 32 n 33 in 2 squares I divide it by 2 to get = 32.5 * 10^4 cells / ml
Is this correct ?? Am not sure
Also when I mix the cell suspension n tryphan blue – I take 100 micro lt of cell susp + 100 micro lt of dye , mix it well n load 10 micro lt onto the hemocytometer .

If I do this type of procedure what sort of dilution factor I have to take into account if any?? Please let me know what to do or is this correct. !! Thanks

-seashell83-

QUOTE (seashell83 @ Jan 25 2008, 11:16 PM)

that is correct. We count all 4 squares so we divide by 4 and then multiply by 10,000.

If you dilute your sample 1:2, then i will multiply the final cell count by 2.

eg. you have counted 50 and 60 in 2 squares.

110/2 = 55 / square

55 x 10,000 x 2 = final cell density.

-scolix-

Hello

I just came across this site which does all the calculations for you if you put in your numbers. Here is the address

http://www.changbioscience.com/cell/hemo.html

I hope it was helpful

Thanks

-dna1977-

dear all..

facing some more curious abt hemacytometer...
if for budding yeast cell, we count the old cell and the new budding cell (they are stick together) as 1 group or jus as 2 cell..??
if counted as 1 group, is it ok if some group got 3 to 4 budding...??
if counted as 2 cell (according to number of cell we get to see), then it regardless of its size??

anyway, thanx a lot for ur help..

-parami-

This protocol describes a method for estimation of mammalian cell number in a defined volume of medium using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted.

--------------------------

reetha.

http://www.protocol-online.org/forums

-reetha-

QUOTE (reetha @ Sep 27 2008, 07:31 PM)

sori... may i know which protocol that u mean?? thanx a lot..

-parami-

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