Simple Isolation of Human Peripheral Blood Monocytes Using Density Gradients of Percoll

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Simple Isolation of Human Peripheral Blood
Monocytes Using Density Gradients of Percoll
 

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Simple Isolation of Human Peripheral Blood
Monocytes Using Density Gradients of Percoll
 

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Introduction (1)

• Monocytes and macrophages play an important role
  in the immune system and are widely used in
  immunological research especially for the study
  of intracellular pathogens, roles of monocytes in
  pathogenesis and generation of dendritic cells
  in vitro.

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Introduction (2)

• Several methods for monocyte isolation from
  peripheral blood are adherence after
  Ficoll-Hypaque purification of peripheral blood
  mononuclear cells (PBMC) and positive and
  negative immuneselection.
• However, monocyte isolation method based on
  density gradient centrifugation is still
  attractive alternative because it is convenient,
  simple and cheap.

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Introduction (3)
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Introduction (4)
Density gradient centrifugation
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Objectives

• To study and establish the method of human blood
  monocyte isolation by using Percoll density
  gradient for an alternative method in our
  laboratory at AFRIMS.
• To determine monocyte purity, viability, yields
  and phagocytosis function after isolation by
  percoll density gradient.

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Methods (1)

• Acid citrate dextrose (ACD) blood from 13 healthy
  blood donors was obtained from blood bank of Army
  Institute of Pathology and blood sample of each
  donor was used in each experiment.
• Two step procedure with single gradient in each
  step for monocytes isolation from whole blood was
  used. First, we used a Ficoll- Hypaque gradient
  (density 1.070 g/ml) for
• seperation of PBMC and then a slight
  hyperosmolar percoll gradient (density 1.064
  g/ml).

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Density gradient centrifugation
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Methods (2)
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Methods (3)
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Methods (4)
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Methods (5)
CD14 Staining and FAC Analysis (Before)
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Methods (6)
CD14 Staining and FAC Analysis (after)
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Methods (7)
Phagocytosis function
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Results (1)

• Table 1. Mean of Monocyte Yields from Flow
  Cytometry Analysis and Viability
• after Ficoll-Hypaque Density
  Gradient
• Mean /-
  SD (range)
• N 13
• PBMC (x106 cells)
  150
• Viability of PBMC
  95.3 /-
  2.6 ( 89 100)
• Monocytes in PBMC
  21.0 /- 6.5 (10 34)
• Monocytes after Percoll gradient
  81.3 /- 4.5 (76 90)
• Viability of monocytes
  94.1 /- 8.1 (70 99)
• Monocytes yield (x106 cells)
  19.3 /- 13.0 (4 39)
• Yield of monocytes
  43.1 /- 18.2 (29 76)
• Phagocytosis of latex beads
  74.8 /- 4.2 (63
  88)

of cells exhibiting latex beads after 24
hours incubation with 5 beads/monocytes
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Results (2)

• Our study demonstrated high percentage of
  viability of both PBMC
• and monocyte determined by trypan blue
  exclusion (95.3 and 94.1,
• respectively).
• The average of percentage of monocytes present
  in the PBMC
• recovered from the initial Ficoll-Hypaque
  gradient was found to be
• 21.0 monocytes.
• From PBMC 150x106 cells, using Percoll gradient,
  an average of
• 19.3x106 monocytes with a purity of 81.3 and
  a recovery of 43.1
• were obtained.
• The functional monocytes detected by
  phagocytosis of latex
• beads was shown to be 74.8 .

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Conclusions

• Percoll density gradient procedure can be done
  with usual
• reagents and equipment of average laboratory.
  Thus, this
• procedure is still attractive alternative
  method because it is
• convenient, simple and cheap although it
  requires more skill and
• time consuming.

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Acknowledgements

• Study Participants
• Armed Forces Research Institute of Medical
  Sciences (AFRIMS)
• COL Narongrid Sirisopana
• COL Thippawan Chuenchitra
• COL Suchitra Sukwit
• LTC Kwanjai Viputtikul
• CAPT Sutchana Tabprasit
• Army Institute of Pathology, Royal Thai Army
  (AIP)
• The Association of Military Surgeons of Thailand

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THANK YOU