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Created page with "= Isolation & Culturing Primary Human Macrophages = Human macrophages can be prepared from anticoagulant-treated peripheral blood mononuclear cells. By culturing mo..."

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= Isolation & Culturing Primary Human Macrophages =

Human macrophages can be prepared from anticoagulant-treated peripheral blood mononuclear cells.  By culturing monocytes with different cytokine cocktails, undifferentiated monocyte-derived macrophages (M0/MDM), M1 polarized or M2 polarized cells can be produced. 

=== Recipes: ===

===== ACD (optional): =====

To 250ml of ddH2O add:

*7.36 g      Citric Acid
*14.71 g    Sodium Citrate
*9.91 g      Dextrose

 

===== Culture Media: =====

*Culture media is RPMI 1640 media supplemented with 10% FBS and antibiotics/antimycotics at the manufacturers recommended concentration.

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=== Protocol: ===

===== Phlebotomy & Mononuclear cell Isolation: =====

#Draw human blood and immediately transfer to a tube containing an appropriate anticoagulant (10ul/ml of 1000U/ml heparin, 1ml ACD/5ml blood, etc).  Mix well.  Draw ~7.5ml of blood for every 12-well plate of macrophages you wish to prepare (e.g. 30ml of blood is sufficient for 4 x 12-well plates).
#Layer blood in 15ml tubes over an equal volume of Lympholyte-poly (Cedarlane) as per manufacturers instructions, being careful to not disturb the interface between the blood and lympholyte-poly.
#Centrifuge at 500 x g for 25 minutes, with the centrifuge acceleration set to 5 and the deceleration set to 0.
#Carefully remove the tubes from the centrifuge and use a pipette to carefully remove the top band of cells (mononuclear fraction).  If needed, the second band (neutrophil fraction) can also be removed.
#Wash isolated mononuclear fraction in room-temperature PBS, 1500 RPM (300G), 6 minutes.  Full acceleration and break can be used for this step.

 

===== Plating Cells: =====

#During the mononuclear cell isolation, add sterile 18mm circular coverglasses to each well of the 12-well plates.  Warm cover-glass loaded plates in the incubator while completing mononuclear cell isolation.
#Suspend the washed mononuclear isolate from step 5, above, in 200ul of culture media per well of cells being plated (e.g. 2.4ml would be used for a single 12-well plate).
#Mix gently, then pipette 200ul of the mononuclear suspension into the centre of each coverglass.  The cell suspension should form a 'bubble' on the coverglass and not be allowed to spread across the whole well.  Adding cells in this fashion will maximize the number of usable cells on each coverglass.
#Carefully transfer to the incubator, and incubate at 37C/5% CO2 for 1 hour.  During this time monocytes will adhere to the coverglasses.
#Remove non-adherent cells (T, B & NK cells) by washing each well twice with 37C PBS.
#Add 1ml of culture media to each well, with the appropriate cytokines added for the desired differentiation state.

 

===== Non-differentiated (M0) Macrophages: =====

#Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
#Culture for 5-7 days, exchanging the media & cytokines if required.

 

===== Classically Activated (M1) Macrophages: =====

#Immediately after step 7 in the 'plating cells' section add 20ng/ml of recombinant human GM-CSF (rhGM-CSF)
#Culture for 5 days, exchanging the media & cytokines if required.
#On day 5 replace media and add 20ng/ml rhGM-CSF + 250ng/ml LPS + 10ng/ml INFγ (*stock is 10,000x).
#Culture an additional 2 days.

 

===== Alternatively Activated (M2) Macrophages: =====

#Immediately after step 7 in the 'plating cells' section add 10ng/ml of recombinant human M-CSF (rhM-CSF)
#Culture for 5 days, exchanging the media & cytokines if required.
#On day 5 replace the media and add 10ng/ml rhM-CSF + 10ng/ml of rhIL-4.
#Culture an additional 2 days.

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