[Cytometry] gentle and non-enzymatic protocols for creating single cell suspensions?

Dr Kim RM Blenman kblenman at cinci.rr.com
Fri Oct 22 16:40:29 EDT 2010


Dear Gelo

 

There are many methods, as you will undoubtedly see from the responses that
you will receive. This is the method that I use

 

For Solid Tissue (spleen, liver, thymus, etc)

1.  Add 5 ml of ice-cold serum free (RPMI or DMEM) medium to a medium size
(60x15mm) polystyrene Petri dish. 

2.  Cut solid tissue of interest into small pieces (~100 mg per piece) and
add to the Petri dish containing the serum-free media.

o    Note: It is best to add no more than 200 mg of tissue to each Petri
dish.

3.  Gently grind tissue between the frosted ends of two frosted microscope
slides while still in the Petri dish. Grind until the tissue is completely
in suspension. Depending on the tissue that you are disrupting there may be
deposits of fat, cartilage, and other “debris” that are resistant to
mechanical disruption. Allow the single cell suspension to sit in the Petri
dish for ~30 seconds to allow the debris to settle and attach to the Petri
dish. Gently swirl the Petri dish then transfer the suspension with a 10 ml
glass pipette into a 15 ml conical tube. Spin for 5 min at 1000 rpm. Discard
medium.

o    Note: At each transfer step, hold pipette attached to Pipet-Aid (or
equivalent system) horizontally and transfer slowly to get rid of large
aggregates (generally debris). The large aggregates will stick to the sides
of the glass pipette.

4.  Add 5-10 ml of Red Blood Cell lysis buffer. Resuspend and incubate at
room temp for 5 min. Add 1 ml horse serum and spin at 1000 rpm for 5 min.
Discard medium.

o    Note: There is some white blood cell death with the addition of Red
Blood Cell lysis buffer. In our hands, the horse serum reduces white blood
cell death caused by addition of Red Blood Cell lysis buffer by providing a
“cushion” between the lysis solution and the white blood cells. 

5.  Wash twice in 5 ml of medium.

6.  Stain as desired

 

For Bone Marrow

1.  Add 5 ml of ice-cold serum free (RPMI or DMEM) medium to a medium size
(60x15mm) polystyrene Petri dish.

2.  Cut off the ends of the bone and place in Petri dish

3.  Remove 2 ml of medium from Petri dish with a syringe then attach a small
gauge needle to the syringe.

4.  Insert needle in one end of the bone and inject medium through the bone
to push bone marrow out of the bone.

5.  Mix bone marrow in medium with a 10 ml pipette to resuspend cells. Then
transfer suspension to a 15 ml conical tube.

6.  Add 5-10 ml of Red Blood Cell lysis buffer. Resuspend and incubate at
room temp for 5 min. Add 1 ml horse serum and spin at 1000 rpm for 5 min.
Discard medium.

7.  Wash twice in 5 ml of medium.

8.  Stain as desired. 

 

Regards

Dr. Kim RM Blenman, CEO

Prospére Center for Immunology Research & Innovation, LLC

Greater Cincinnati Ohio Area

USA

Ph #: 513-234-9140

Fax #: 513-234-9140

E-mail: kblenman at cinci.rr.com

Website: www.ProspereImmunology.com

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-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Gelo de la Cruz
Sent: Monday, October 18, 2010 8:54 PM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] gentle and non-enzymatic protocols for creating single
cell suspensions?

 

Hi Flow-ers.

 

I have a researcher wanting to study the cell surface proteome on spleen,

bone marrow, etc. using flow cytometry.  We are trying to find protocols on

creating single cell suspensions that are not very harsh and do not involve

trypsin, as we want to preserve cell surface proteins.  I believe

collagenases might work, but have no solid leads.

 

Does anyone have any experience in this?  Suggestions?  Protocols?

 

Thank you very much!

 

 

 

Gelo

 

-----------

DKFZ/HI-STEM

Heidelberg, Germany

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