1. Chapter 11. 원심분리를 이용한 마우스 혈액 면역 세포 분리
담당 교수: 하상준 교수님
담당 조교: 안상용
과학원 S405

2. Blood
Qualifies as a type of connective tissue
- has cells and a significant extracellular component
Functions:
- Transport of nutrients and oxygen to the rest of the body
- Transport of wastes and carbon dioxide away from cells and tissues of
the body
- Transport of hormones
- Thermoregulation (homeostasis)
- Transport of leukocytes to sites of immune reaction

3. Blood
Divided into:
- Plasma (~55% of blood volume)
• Mostly water
- ~90% of plasma (by weight)
• Protein
- albumin
- globulins – immunoglobulins, etc
- fibrinogen
• Nutrients, hormones, electrolytes
- “Formed Elements” (~45% of blood volume)

4. 면역장기 및 면역세포의 종류
Primary lymphoid organs
- Involved in lymphocyte development and “education”
- Bone marrow, thymus
Secondary lymphoid organs
- Contain immunocompetent cells that can undergo antigen-dependent
activation
- Lymph nodes, spleen, tonsils, adenoids, peyer’s patches, appendix

5. 면역장기 및 면역세포의 종류
Erythrocytes
Leukocytes
- Granulocytes
• Neutrophils
• Eosinophils
• Basophils
- Agranulocytes
• Monocytes
• Lymphocytes
Platelets

6. 원심분리 (Centrifugation)
Uses a centrifugal force to separate mixtures used in industry and in
laboratory settings
The rate of centrifugation can be expressed as g or rpm
Types of centrifugation:
- Differential centrifugation
- Density gradient centrifugation

7. 원심분리 (Centrifugation)
Differential centrifugation
- A procedure in which the homogenate is subjected to repeated
centrifugations each time increasing the centrifugal force
- Separation due to size difference

8. 원심분리 (Centrifugation)
Density gradient centrifugation
- Used to isolate nucleic acid and organelles via buoyant density based on
amounts of lipid, protein or nucleic acid
- Different specific gravity causes floating at different levels in density
gradient
• Rate-zonal centrifugation
• Isopycnic centrifugation

9. 원심분리 (Centrifugation)
Rate-zonal centrifugation
- “Velocity sedimentation”
- The sample is layered on the top of a density gradient
- Under centrifugal force, the particles sediment through the gradient in
separate zones according to their size, shape and density
Isopycnic centrifugation
- “Equilibrium sedimentation”
- The sample is uniformly mixed with the gradient material
- Each of the particles sediments only to the position at which the gradient
density is equal to its own density

10. 원심분리 (Centrifugation)
velocity sedimentation over sucrose
equilibrium sedimentation:
sucrose or CsCl

11. Types of Centrifuges
Low-speed centrifuge
- “Table centrifuge”
- Max. speed 6,000 rpm (6,000 xg)
- Cell, nucleus (easily precipitated material)
High-speed centrifuge
- Max. speed 20,000-25,000 rpm (60,000 xg)
- Cell, nucleus, organelles
Ultracentrifuge
- Max. speed 40,000-80,000 rpm (600,000 xg)
- Cell, nucleus, organelles, components of cell membrane, ribosome,
polysome, macromolecules

12. Types of Centrifuge Rotors
Fixed angle rotor
Swinging bucket rotor
Vertical rotor

13. PBMC (Peripheral Blood Mononuclear Cell) Isolation using Histopaque
Histopaque-1077 is a polysaccharide gradient solution designed for cell
separation techniques
- Density = 1.077±0.001
mostly lymphocytes

14. Cell Counting

15. Live Cell Counting using Trypan Blue Exclusion
Count the number of live cells by excluding cells stained with trypan blue
- Dead cells easily uptake trypan blue dye due to their weakened
membrane
- Cell viability = (# live cells / total # cells) x 100%

16. Experimental Procedures
Isolation of PBMC
1) 증류수에 녹인 4% sodium citrate 0.5mL이 담긴 tube를 전자저울로 무게를
잰다.
2) 흡입 마취제로 마우스를 마취시킨 뒤 모세관을 이용해 마우스 안구에서 약
3-4 drops의 혈액을 채취하여 위의 tube에 담는다.
3) 마우스 혈액이 채취된 tube를 다시 전자저울로 무게를 재고 채취된 혈액의
무게를 계산한다.
4) 혈액을 채취한 tube에 2% RPMI 1.5mL을 넣어준다.
5) Vortex를 이용해 tube에 든 sample을 섞어준다.
6) Pasteur pipette을 sample tube에 꽂고, histopaque-1077 solution 1.5mL을
주입한다.
7) 원심분리기를 2,000rpm, 20min, 20℃ with no brake로 설정하고, sample
tube를 원심분리 시켜준다.

17. Experimental Procedures
8) 2% RPMI 1.5mL이 담긴 새로운 tube에 원심분리 시킨 PBMC를 스포이드
고무를 장착시킨 pasteur pipette을 이용해 옮겨준다.
9) 원심분리기를 2,000rpm, 10min, 4℃ with brake로 설정하고, sample tube
를 원심분리 시켜준다.
10) 원심분리 후 상층액은 버리고 남은 pellet을 얻는다.
11) Pellet을 0.5mL의 2% RPMI로 resuspension 한다.
Counting live PBMC
1) Trypan blue로 분리한 mononuclear cell suspension을 2배 희석 시킨다.
(50uL + 50uL)
2) Pipette을 이용해 샘플 10uL를 유리 챔버와 커버 유리 사이에 주입한다.
3) 푸른색으로 염색되지 않은 살아있는 세포의 수를 센다.

18. Further Study
1) Explain the difference between g-force and rpm in centrifugation
2) Why the obtained mouse blood is added to sodium citrate immediately?
3) What can be a possible reason if your PBMC viability is very low?

19. Lab Report Writing
실험 보고서 양식대로 작성 (p.3)
- 결과:
• 채취한 혈액의 무게
• 매뉴얼에 제시된 3문제들에 대한 답 (Include equations)
- Blood density ~1.060 g/cm3
- 고찰:
• Further study 참조
Hand writing only
- 컴퓨터 typing시 0점 처리
실험 종료 후 일주일 이내로 작성하여 S405호 제출 (3pm 까지)
- 지각제출 감점