HiPer® Lymphocyte Separation Teaching Kit
Product Code: HTI010
Number of experiments that can be performed: 5
Duration of Experiment:
Protocol: 2 hours
Storage Instructions:
The kit is stable for 6 months from the date of receipt
Store Lymphocyte Separation Media at 2-8oC
Other reagents can be stored at room temperature (15-25oC)
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Index
Sr. No.
Contents
Page No.
1
Aim
3
2
Introduction
3
3
Principle
3
4
Kit Contents
4
5
Materials Required But Not Provided
5
6
Storage
5
7
Important Instructions
5
8
Procedure
5
9
Observation and Result
6
10
Interpretation
6
11
Troubleshooting Guide
7
2
Aim:
To separate lymphocytes from human blood using density gradient media.
Introduction:
The Lymphocyte Separation Medium (LSM) is an iso-osmotic, low viscosity medium containing polysucrose
and sodium diatrizoate adjusted to a density of 1.0770 +/- 0.0010 g/ml. This medium offers a quick and
reliable method for the simple isolation of human mononuclear cells and lymphocytes from defibrinated EDTA
human blood.
Principle:
White blood cells (WBCs), or leukocytes (also spelled "leucocytes"), are cells of the immune system involved
in defending the body against both infectious disease and foreign materials. Five different and diverse types of
leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a
hematopoietic stem cell. Leukocytes are found throughout the body, including the blood and lymphatic
system. There are several different types of white blood cells. They all have many things in common, but are
all distinct in form and function. A major distinguishing feature of some leukocytes is the presence of
granules; white blood cells are often characterized as granulocytes or agranulocytes as shown in the
following table:
Microscopic
appearance
Type
Diagram
Nucleus
Neutrophil
Multilobed
Eosinophil
Bi-lobed
Granulocytes
Basophil
Bi-lobed or
tri-lobed
Lymphocyte
Eccentric
Agranulocytes
Monocyte
Kidney bean
shaped
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The first step in studying lymphocytes is to isolate them so that their behavior can be analyzed in vitro.
Lymphocytes are present in blood, peritoneal exudates or lymphoid organs mixed with other cells. Human
lymphocytes can be isolated most readily from peripheral blood. A pure population of lymphocytes can be
obtained by various separation procedures. Isolation of lymphocytes is based on the adapted centrifugation
method of Boyum in which diluted defibrinated blood is layered on a solution of sodium diatrizoate and
polysucrose and centrifuged at low speeds for 30 minutes. Differential migration following centrifugation
results in the formation of several cell layers. Mononuclear cells (lymphocytes and monocytes) and platelets
are contained in the banded plasma-LSM interphase due to their density, and the pellet that is formed
contains mostly erythrocytes and granulocytes, which have migrated through the gradient to the bottom of
the tube. Most extraneous platelets are removed by low speed centrifugation during the washing steps.
Lymphocytes or other mononuclear cells (monocytes or mesenchymal stromal cells) or granulocytes are
recovered by aspirating the plasma layer and then removing the cells. Excess platelets, LSM and plasma can
then be removed by cell washing with isotonic diluent buffer.
(Buffy coat)
Fig1: After differential centrifugation lymphocytes and monocytes are found in the buffy coat.
Kit Contents:
HiPer® Lymphocyte Separation Teaching Kit enables separation of lymphocyte by using density gradient
media.
Table 1: Enlists the materials provided in this kit with their quantity and recommended storage
Sr.
No.
1
2
3
4
5
6
7
Product
Code
LS001
PW144
CG281
TKC219
S011
RM9971
TCL005
Materials Provided
HiSep LSM 1077
Centrifuge Tube (15 ml)
Polypropylene Tube (0.5 ml)
Diluent Buffer
Giemsa’s Stain
Cedar wood Oil
Trypan Blue (0.5%)
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Quantity
5 expts
15 ml
6 Nos.
6 Nos.
60 ml
3 ml
0.3 ml
0.3 ml
Storage
2-8oC
RT
RT
2-8oC
RT
RT
RT
Materials Required But Not Provided:
Reagents: 70% Alcohol/ Spirit, Methanol
Other requirements: EDTA/Heparin coated collection tube, Cotton, Glass pasteur pipettes, Hemocytometer,
Microscopic slides, Coverslips, Micropipettes & Tips
Storage:
HiPer® Lymphocyte Separation Teaching Kit is stable for 6 months from the date of receipt without showing
any reduction in performance. On receipt, store the HiSep LSM 1077 and Diluent Buffer at 2-8oC. Other
contents can be stored at room temperature (15-25oC)
Important Instructions:
1.
Before starting the experiment the entire procedure has to be read carefully.
2.
Always use fresh anticoagulated blood while performing the experiment.
3.
Set the brake of the centrifuge machine to OFF mode.
4.
Perform the experiment within 2 hours of blood collection.
Procedure:
Lymphocyte Layer Separation and viability count:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Collect 4 ml of blood in the EDTA/Heparin coated collection tube, using sterile syringe and needle.
Mix immediately by inverting or vigorously shaking the tube for EDTA to be uniformly distributed.
Dilute the blood by adding 4 ml of diluent buffer.
Take 2.5 ml of HiSep LSM 1077 in a new 15 ml centrifuge tube. Overlay the LSM with 7.5 ml of
diluted blood.
Centrifuge at 2300 rpm for 30 minutes in a fixed angle rotor. (If swinging bucket rotor is used
centrifuge at 1600 rpm).
NOTE: Always keep the brake off during centrifugation.
Using a clean glass pasteur pipette carefully remove the lymphocyte layer in a new collection tube.
Add 5 ml of diluent buffer to the lymphocyte layer. Mix by gentle pipetting and centrifuge at 1900
rpm for 10 minutes
NOTE: This step helps to reduce the number of platelets.
Discard the supernatant obtained from above step.
Resuspend the pellet in 500 µl of diluent buffer.
Take 10 µl from above step in new collection tube and add 40 µl of diluent buffer and 50 µl of Trypan
Blue.
Place a coverslip on the Neubauer chamber of the haemocytometer.
Cover one side of the Neubauer chamber of the haemocytometer with the sample.
Observe and count the live and dead cells under 45X magnification in a light microscope.
Differential Staining:
1.
2.
Spin the tube from step 9 at 2300 rpm for 10 minutes.
Discard the entire supernatant leaving around 50 µl in the tube.
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3.
4.
5.
6.
7.
8.
Resuspend the pellet in the same solution and make a smear on a microscopic slide.
Air dry the slide for 5-10 minutes.
Add 5-10 ml of methanol to cover the slide. This step helps in fixing the cells onto the slide.
Discard the methanol carefully.
Add 15 ml of staining solution and incubate for 30 minutes at room temperature.
Observe under oil emulsion lens and look for the lymphocytes and monocytes.
Observation and Result:
Count the viable and dead cells under the microscope (in four WBC chambers of the haemocytometer) after
trypan blue staining and record your data as in the following table:
Viable cells (cells without dye)
Dead cells (cells with dye)
Calculate the number of total cells as follows:
Area of one WBC chamber = L X H
1mm X 1mm
= 1mm2
Depth of Haemocytometer is 0.1mm
Hence Volume of 1 WBC chamber = Area X Depth
= 1mm X 0.1mm
= 0.1mm3
If 4 WBC chambers are counted then volume of 4 WBC chambers is 4 X 0.1= 0.4mm3
Total no. of cells (cells/ml) =
Total cells_
X 1000 X Dilution Factor
Volume of WBC chamber
=
=
Total cells
X 1000 X 5
0.4mm3
cells / ml
Count the percentage of isolated lymphocytes, monocytes and total granulocytes under the microscope after
performing the differential staining and record it as in the following table:
No. of total
cells
No. of viable cells
Lymphocytes
(%)
Monocytes
(%)
Granulocytes
(%)
Interpretation:
After performing the lymphocyte separation procedure by using LSM, viable lymphocytes are isolated
predominantly compared to granulocytes and monocytes.
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Troubleshooting Guide:
Sr.No.
Problem
Possible Cause
Solution
1
Buffy coat is not
formed
Blood sample is not of good
quality
Blood should be collected asceptically in the
presence of EDTA or Heparin and should be
processed within 2 hours of collection
2
RBC contamination
in the buffy coat
Washing is not done
properly
Follow the washing steps exactly as mentioned
3
Higher percentage
of dead cells
Cell counting was not done
immediately after trypan
blue staining
Cell counting should be done immediately after
doing the trypan blue staining
Technical Assistance:
At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of
technical assistance mail at [email protected]
PIHTI010_O/0514
HTI010-03
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