Chapter 11. 원심분리를 이용한 마우스 혈액 면역 세포 분리 담당 교수: 하상준 교수님 담당 조교: 안상용 ahnsy221@gmail.com 과학원 S405

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

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)

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

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

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

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

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

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

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

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

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

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

Cell Counting

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%

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.

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.

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?

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