MACROPHAGE ISOLATION AND CULTURE FROM WHOLE BLOOD

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neno
neno's picture
MACROPHAGE ISOLATION AND CULTURE FROM WHOLE BLOOD

HI !
I am trying to isolate and culture macrophages from whole blood. wht is the appropriate centifuge speed and criteria reqiured for buffy coat isolation using histopaque(sigma) or ficoll.
also amount of RPMI medium require for mononuclear cell(6-15*10e6)
can anybody help me. i m trying first time on cell culture.
manisha

g a
g a's picture
Hi Manisha

Hi Manisha
Ficoll gradient centrugation is a highly practiced procedure for isolation of granulocytes.
check this link for the protocol.
http://www.miltenyibiotec.com/download/protocols_sample_preparation_en/496/SP_granulocytes_PB_density_gradient.pdf

neno
neno's picture
thanks

thanks
any idea for counting and staining of mononuclear cell which i have isolated from buffu coat?
how much RPMI media require for resuspend the cell pellet of MNCs derived from buffy coat?
i m trying with 20 ml blood initially.
 

g a
g a's picture
Hi Manisha

Hi Manisha
For counting the mononuclear cells you can use the haemocytometer.
You can stain your cells with trypan blue for assessment of viability and with giemsa to check the differential leucocyte count.
1ml of final suspension should be enough for making MNCs from 20ml blood.
But you will need to seed at cell density recommended for the culture of MNCs. You can refer to Freshney for detailed explanation.
All the Best

neno
neno's picture
thank u sir. when i  m

thank u sir. when i  m staining MNC ,some lymphocytes also seen. and i require only pure culture of mnc(macrophage).heamocytometer nt counts only MNC,so how i know quantity of mnc .

g a
g a's picture
Hi Manisha

Hi Manisha
I dont know exact method for the counting of Macrophages from blood, however I can give you a logical method that I hope will work out for you.
Calculate the total No of cells in your pellet and perform the differential count of the same pellet identifying the nucleus like multilobed are neutrophils, round cells are lymphocytes etc.............. and then calculate the % of the cells contributed by lymphocytes and subtract that from total count which will be your answer.
Alternatively, If your goal is to culture these cells, then you seed the total cells and later mildly wash with the medium oor PBS, since lymphiocytes dont attach to the culture vessels they are aspirated and now you trypsinize your attached cells and count the same.

MUTHUMARIAPPAN ...
MUTHUMARIAPPAN Murugan's picture
Dear Manisha,
R Bishop
R Bishop's picture
You might read through this
neno
neno's picture
Hi

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.