Chemokine Receptors in Whole Blood

Hello!

I am a PhD student and trying to do a flow cytometry experiment to see the
expression level of chemokine receptors in peripheral blood. I have a few
questions in this regard and help would be appreciated. The questions are as
follows:

1. For how long and what temperature ideally I should be incubating the
antibodies for ? I have done 2 experiments by incubating the antibodies at
room temperature but have been told that at room temperature, chemokine
receptors may internalise and so I won't be getting the reliable data, is it
true ?

2. I am lysing the whole blood with lysing buffer and the current protocol I
have suggest to add the antibody, blood and lysing buffer at the same time.
However, literature and the lysing buffer data sheet talks about lysing the
blood after incubation with antibodies. I am just wondering that if there
is any idata/information on the difference in lysing the blood with
incubating antibodies or followning the antibody incubation. I would also be
interested to know that how much lysing buffer should be used ? I have
looked at many papers but I don't think that there is any hard and fast rule
for this,  people are using 2ml lysis buffer for 50ul blood, however the BD
Bioscience suggests to use 2ml for 200ul blood, so for 50ul of blood I
should be using 500ul of lysing buffer, any comments ?

3. I will be analysing my cells on FACS machine within 1-4 hrs after
preparation, do I still need to fix my cells as I dont want to store them
for longer ?

4. I am hoping to see the chemokine receptors expression on lymphocytes,
monocytes and granulocytes, can I use a single template and gate all the
three sub-populations OR do I have to make individual tempelate for each
cell population ?

5. Can anyone suggests a good marker for neutrophils ? I have been told
that CD14-CD11b+ cells are definitely neutrophils, any comments ?

6. On a separate note, I am also interested to look at the chemokine
receptors expression on mast cells using anti-c-kit (CD117), could you
please suggest me how to gate the cells in order to see the c-kit positive
cells ?

I would be grateful if anyone could help me with this, please feel free to
ask any further details if I haven't made things very clear. I will look
forward to hearing soon.


Best wishes,

Abdul
Glasgow