1. Tissue Sample Preparation

1. Tissue Sample Preparation
1.1 Isolation of mononuclear cells from human peripheral blood or bone marrow
aspirates
Reagents
1.
Histopaque-1077 (Sigma # 1077-1)
2.
Phosphate Buffered Saline (PBS)
Procedure
1. To a 15-mL conical centrifuge tube, add 5.0 mL Histopaque-1077 and bring to room
temperature.
2. Prepare specimen mixtures in separate 15-mL tube: Mix 5 mL of blood/bone marrow
aspirate with 5 mL of PBS.
3. Delicately overlay blood/bone marrow over to Histopaque. Centrifuge at 400 x g for 20
minutes at room temperature with no brake.
4. The mononuclear layer is visible as a white cloudy layer situated at the interface
between the two solutions. Carefully aspirate the interface with a pasteur pipette
and transfer to another 15-mL tube.
5. Fill this tube with PBS and centrifuge at 500 x g for 5 minutes.
6. Aspirate the supernatant and discard.
7. Resuspend cell pellet with 5.0 mL PBS and repeat steps 5 and 6.
8. Discard supernatant and resuspend cell pellet in 1 mL of PBS.
1.2 Neutrophil isolation from human peripheral blood
Reagents
Histopaque 1119 (Sigma #1119-1)
Histopaque 1077 (Sigma 1077-1)
Phosphate Buffered Saline Solution (PBS)
4. Add 3 mL Histopaque 1119 to a 15-mL conical centrifuge tube.
5. Carefully layer 3 mL of Histopaque 1077 onto the Histopaque 1119.
6. Carefully layer 6 mL of whole blood onto the upper gradient of the tube from step 2.
7. Centrifuge at 700 x g for 30 minutes at room temperature.
8. Carefully remove centrifuge tubes. Two distinct opaque layers should be observed.
9. Aspirate and discard fluid to within 0.5 cm of layer A. Transfer cells from this layer to a
Box 43131 | Lubbock, Texas 79409-3131
Updated May 2012
Phone – 806-742-2715
1
tube marked "mononuclear cells".
10.
Aspirate and discard remaining fluid to within 0.5 cm of layer B. Transfer cells
from this layer to a tube labeled "granulocytes".
11.
Wash the cells by addition of 10 mL PBS. Centrifuge 10 minutes at 500 x g.
Remove the supernatant and discard.
12.
Resuspend the cells by gentle aspiration with a Pasteur pipette.
13.
Repeat steps 8 and 9.
14.
Resuspend cells in an appropriate volume of PBS and use as desired.
1.3 Isolation of total white cells from human peripheral blood by red cell lysis
Non-fixing lysing solution
Ammonium Chloride Lyse
10X concentration
Dilute 10 mL stock with 90 mL double distilled water. Store at 4°C.
NH4Cl (ammonium chloride) 80.2 g
NaHCO3 (sodium bicarbonate) 8.4 g
EDTA (disodium) 3.7 g
Working solution
QS to 1 liter with double distilled water. Store stock at 4°C for up to six months.
Procedure
1. Obtain a whole specimen in a 10-mL heparinized tube, preferably preservative free.
2. Aliquot 2 mL into each of four 50-mL conical centrifuge tubes.
3. Immediately fill tubes up to 50 mL with cold 1X lysing solution.
4. Rock for 10 minutes at room temperature.
5. Spin down at 4°C for 10 minutes at 250 x g.
6. Decant supernatant and allow tubes to drain briefly.
7. Resuspend pellet by drawing tube across tube rack (i.e., scrape the pellet).
8. Combine two pellets into one 50-mL tube. Fill to 10 mL with cold PBS to wash.
9. Spin, decant and scrape pellet as above.
10.
Combine to the two pellets into one tube and wash with 10 mL cold PBS.
11.
Spin, decant and scrape pellet. Use cells as needed.
Note: This osmotic lysis method does not harm the white cells or alter membrane
asymmetry. Due to the osmotic method of lysis, the 10X stock must be diluted to 1X
Box 43131 | Lubbock, Texas 79409-3131
Updated May 2012
Phone – 806-742-2715
2
with water. Using a buffer such as PBS alters the osmolarity of the lysis buffer and it will
no longer be effective. This lysis technique will leave all populations of white cells,
including granulocytes.
Further Reading: Modified from the Handbook of Flow Cytometry Methods, J. Paul
Robinson, editor, Wiley-Liss Inc.
1.4 Preparation of single cells from solid tissues
A common feature of all flow cytometric studies of solid tissues is the requirement from
dispersal into single cells (or nuclei) before staining and analysis. Dispersal of solid
tumors for flow cytometric analysis can be achieved by a variety of methods, most of
which employ a combination of mechanical and enzymatic disaggregation. The choice
of dispersal method depends on the type of material to be examined and the point to be
measured. Also, special treatment is required for:
1.5 Cell Counts (Hemacytometer)
Hemacytometer grid system
Errors can be introduced in a number of ways: dilution errors, loss of cells during
pipetting, uneven suspension of cells, overfilling or underfilling of the chamber and the
counting of cells before they settle. Random distribution of cells in the chamber is
another source of error but can be compensated for by counting a large number of cells.
Clean chamber just before and just after use. If the chamber is loaded too heavily, clean
it and begin again; do not attempt to remove excess liquid.
Example: Sample of cell suspension diluted 1:1 with trypan blue
Grid #1 (16 squares)= 195 cells
Grid #2 (16 squares)= 177 cells
Grid #3 (16 squares)= 204 cells
Grid #4 (16 squares)= 166 cells
total = 742 cells
mean = 185.5 cells
Formula:
Cell concentration (cells/mL)= mean x (10) x dilution factor
= 185.5 x (104) x 2
= 3.71 x 106 cells/mL
1.6 Thawing Frozen Cells
1. Quickly thaw frozen vial of cells by brief incubation of tube at 37°C in a water bath
until sample is just slushy. Wipe tube well with 70% ethanol.
2. Transfer sample to 15-mL conical tube containing 10-13 mL RPMI 1640 complete
medium warmed to 37°C.
3. Centrifuge cells immediately at 800 x g. Remove supernatant.
Box 43131 | Lubbock, Texas 79409-3131
Updated May 2012
Phone – 806-742-2715
3
4. Wash cells 2X with 10 mL RPMI complete. Centrifuge cells.
5. Resuspend cells in desired buffer or medium and use as needed.
Notes: DMSO is a polar planar molecule which is not only a differentiating agent but is
also toxic to the cells. This toxicity is marked at warm temperatures. It is very important,
therefore, to remove the cells from the freezing medium as rapidly as possible. This is
accomplished by diluting the thawed cell suspension immediately into a large volume of
medium and centrifuging the cells to remove the diluted DMSO.
1.7 Freezing Samples and Cell Lines
1. Either lyse RBCs or Ficoll cells using sterile methods. Wash cells 1X with cold sterile
medium.
2. Resuspend cells in an amount of cold medium. Count cells. Centrifuge sample.
3. Cells should be frozen in aliquots of 10-20 x 106 cells. Resuspend pellet in 500 µL of
cold medium per tube.
4. Label cryotubes with appropriate information: Cell Lines: Name of cell
line Date Passage number (if known) Source of cells Number of cells Primary
tissue: Last name and first initial of patient MD Anderson
number Date Diagnosis Tissue type (BM, PB, Ph) Treatment (F= ficolled, L=
lysed)
5. Add an equal amount of freezing medium (recipe following) to the RPMI drop-wise
while gently swirling. The ratio of RPMI cell suspension to freezing medium
should always be 1:1.
6. Add 1 mL of the resulting cell suspension to the appropriate crytubes. Put tubes on
ice.
For freezing in the -70°C freezer
1. Place tubes in a cryobox which has been cooled to 4°C.
2. Place cryobox into -70°C freezer.
3. Remove frozen vials the next day to liquid nitrogen freezer.
4. Store empty cryobox in refrigerator.
5. For freezing in controlled freezer:
You may need a pilot tube as an internal control for the degreed freezer.
In a cryovial place 500 µL RPMI and 500 µL freezing medium. Put on ice
with prepared tubes.
Cool the degreed freezer chamber to 4°C.
Place cryovials into chamber. The freezing process takes about an hour.
Remove tubes immediately to liquid nitrogen freezer.
Freezing Medium
Box 43131 | Lubbock, Texas 79409-3131
Updated May 2012
Phone – 806-742-2715
4
40 mL Hank's Buffered Saline Solution (HBSS) - Ca+2, -Mg+2
20 mL DMSO
40 mL fetal calf serum
100 mL
Notes: DMSO is a polar planar molecule which is not only a differentiating agent but is
also toxic to the cells. This toxicity is marked at warm temperatures. It is very important
therefore, to add the DMSO as the last step, keep cells on ice once DMSO is added,
and start the freezing process as soon as possible. DMSO keeps cells viable during
freezing by preventing the water within cells from crystallizing and bursting the lipid
membrane.
Box 43131 | Lubbock, Texas 79409-3131
Updated May 2012
Phone – 806-742-2715
5