cell counting

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neno
neno's picture
cell counting

 

Hi Can u help me for counting cells by use of neubars chamber. I am currently working on a protocol on culture of macrophage from whole blood. It is something like this:(i m initially deal with 20 ml blood for practice) From the 20 ml blood, buffy coat is removed.(800*g,15 min.,R.T) then PBS is added (1:2) Overlay on the histopaque (1.083)(sigma) at equal amount. centrifuge.(800*g,15 min.,R.T) MNC ring is collected. Add 3 volume of PBS and centrifuge with 250 G for 10 minutes, RT. supernant removed and pellet collected. (2 times wash). now, i resuspend pellet by adding 4 ml PBS/ MEDIA. then again 1:20 dilution is done by using WBC diluting fluied with thoma pipette(white bid) and by neubars chamber i got 107 cells (counting 4 WBC quaters) now,wht is the my cell concentration in pellet? I got result variation by using different formula. Can u help me ?(according to my calculation it is around 21*10^6, is it true?) There is an issue about contamination RBCs, so how to get rid of them? thanks.
 

 
 

heehawmcduff
heehawmcduff's picture
That's a pretty good number

That's a pretty good number of monos from 20ml whole blood.  I would expect a cell number of around 10 million so if you are getting 20 million then that's ok.
 
Can you remove the red cells with hypotonic lysis?

samm
samm's picture
The formula for using the WBC

The formula for using the WBC chamber (4x4 grid) using 10 ul of sample on a hemocytometer is:
[(Count 1+2+3+4)/4] x 10,000 x final dilution factor = # cells/ml
Each count includes the entire 4x4 grid, which has 16 small squares. Final df includes all previous dilutions - e.g. 1:10 from buffy coat pellet further diluted 1:5 in trypan blue results in df of 50.
Count range is ideally 20-300 per 4x4 grid.
You can easily eliminate most RBCs in your prep using Gey's solution/AcK lysis.
 

Guy Sovak
Guy Sovak's picture
Hi,

Hi,
Look at this web site and make sure that you have the same chamber pattern.
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html
Guy

Abhishek Singh
Abhishek Singh's picture
there are various questions

there are various questions
i am answering how to count in neubar chamber
first locate central chamber containing five triple line enclose squares covered by horizontal and vertical arrangenment of triple line .
place cell suspension near corner of slide so that suspension goes through capillary action
locate refractive rings in visual field and then start count count 100-300 cells and number of square counted smallest square is 50μmx50μm and height is 0.1mm so find out volume and then multiply by dilution factor 10^4

neno
neno's picture
thanks.

thanks.
now,there are some problem with my mononuclear cell culture.i found some very tiny,moving(like vibratting) black round things with my 35 mm cell culture plate. i had crosschecked them on N-agar plate,blood agar plate. but nothing contamination was there.but when i gramstained 10 microliter cell culture ,there was a cocci liked cells. i don’t understand this unidentified things. and i want to get rid of them really. is it contamination or platelets r there?please help me .
 

20101975
20101975's picture
responding to your last

responding to your last question, i would suggest you to attach few photgrapfs of plates so that one can suggest you something.

neno
neno's picture
now, another thing that i

now, another thing that i want to ask u that is how to scrap the cell from culture flask using cell scraper and what to do next with then for staining with ANAE(SIGMA). we have not facility of slide centrifuge with us. any alternative?
i mean how to attech the cell on the slide for cell staining?plz answer me soon. i'll send culture pics on next reply coz i have some problem with my laptop.
thanks.