Abhishek Kumar Mishra

1. Separation of Peripheral
Blood Mononuclear Cells
TEACHER:
Dr. Gurjeet Kaur
Subject:
Good lab practices and
Instrumentation
Presented by:
Ritesh pandey A7104421012
Aryan Shukla A7104421014
Nikhil Singh A7104421016
Om tripathi A7104421006
Abhishek Mishra A7104421010
Rishabh Mishra A7104421034

2. What is Centrifuge?
• A centrifuge is a device used to separate components of a mixture
on the basis of their size, density, the viscosity of the medium, and
the rotor speed.
• In a centrifuge, the sample is kept in a rotor that is rotated about a
fixed point, resulting in strong force perpendicular to the axis.
Centrifugation
• Centrifugation is a method of separating molecules having different
densities by spinning them in solution around an axis at high speed
• Centrifugation is used to collect cells, to precipitate DNA, to purify
virus particles.

3. What is HiSep Lsm
• HiSep Lsm 1073 is based on the adapted method of isolating
lower density mononuclear cells using centrifugation
techniques.
• HiSep TM LSM 1073 is an iso‐osmotic, low viscosity medium
containing polysucrose and sodium diatrizoate, adjusted to a
density of 1.073± 0.001 g/ml.
• This medium offers a quick and reliable method for the simple
isolation of human mononuclear cells and lymphocytes.
• The method is applicable for studying cell-mediated
lymphocytes and for human lympholysis and for human
lymphocyte antigen (HLA) typing.
• It may be employed as the initial step prior to enumeration of T,
B, and “null” lymphocytes.

4. Precautions before using HiSep Lsm
• For in vitro Diagnostic use.
• Dilution or adulteration of this reagent may result in inadequate
mononuclear cells separation.
• Do not use reagent beyond expiry date.
• The solution may cause sensitization by inhalation and skin
contact. Wear suitable protective clothing and gloves.
• Never pipette by mouth and avoid contact with skin and
mucous membranes.
• Avoid microbial contamination of reagents, which may lead to
incorrect results.

5. Procedure
1. 2.5 ml HiSep Lsm taken in a fresh tube.
2. Whole blood (4.0 ml) was taken in a tube with 0.5ml 3.8%
Trisodium Citrate .
3. Then mix it gently.
4. Dilute the 4.5 ml blood with 4.5 ml of 1xPBS.
5. Overlay gently 9.0ml diluted blood on HiSep Lsm.
6. Centrifuge the sample at 8°C at 1200/1500 rpm.
7. For 15 minutes with no brakes off.

6. • Result
Separate all the cells in different tubes for further studies or
isolation of homogenous cells.

7. Application
• Isolation of lower density mononuclear cells for e.g-
Mesenchymal stromal cells or monocytes.
• Separation of human peripheral blood .
Disposal
• User must ensure proper cleaning of equipment and floors with
plenty of water. Offer surplus and non‐recyclable solutions to a
licensed disposal company.

8. MCQS Based on the Practical
Q1. Buffy coat consists of?
A) WBCs and platelets C) WBCs and
factors
B) B) WBCs only D) peripheral blood
mononucleus cells
Q2. What is in hisep LSM?
A) anhydrous sodium diatrizoate+ simple sucrose+ buffer
B) B)sodium diatrizoate hydrate
C) C) polysucrose D)all of the above
E)only B&C

9. Q3. Trypan blue is used for
A) Staining dead cells to determine cell viability.
B) Staining lymphocytes.
Q4. Why is hisep LSM 1073 kept in tight bottle?
A) It's volatile B) vapours can cause slow
damage to cells
C) It's light sensitive D) It can absorb atmospheric
water and become dilute
E) All of the above except D
Q5. Why fresh blood is used for this experiment?
A) To ensure good separation and high viability of isolated cells.
B) To ensure quick results within seconds.