Lab 3 Cell Biology 2015, Kristina Ruuth
Characterisation of novel anticancer drugs PART 2
• Most currently used anticancer drugs
work by targeting the cytoskeleton,
cell cycle progression or the
apoptotic machinery.
• Tumor resistance to anti-cancer drugs
Ref Per Holmfeldt/Mikael Sellin, Umeå University
Lab3: Each group will test 7 substanses
from a chemical library plus 1 solvent
control on Jurkat cells
Cell viabilty:
1. Tryptan blue exclusion after 24 and 48 h.
2. Metabolic activity: Cell mediated
reduction of resazurin after 24 h.
Giemsa stain.
FACS analysis:
Cells double stained with immuno
fluorescence technique for micro tubules
and PI for cell cycle status.
Dipak M et al
Nature Comm
2013
Cell viabilty:
Trypan blue exclusion: Counting of trypan blue stained cells in a burker chamber.
Count all samples in triplicate at 2 time points ,
24 h and 48h after start of treatment.
Triplicate: Count viable cells in at least 3 “A-squares”
calculate average . Repeat this twice for each
treatment and the solvent control.
Cell conc 0.5x106 cell/ml
Diluted 1:2 will result in approx 25 cells/A-Square with
large fluctuations.
Hints: Dilute 4:5 , eg 200uL cells + 50 uL Trypanblue .
Take out samples for counting and place the tubes on
ice. Storage on ice for a couple of hours will not affect
cell viability.
Cell Viability
Metabolic activity, Proliferation assay.
Many assays: MTT, WST, 3H-thymidine, Brdu incorporation, Resazurin.
Resazurin (Alamar Blue):
Untreated cells +
resazurin
Medium +
resazurin
Analyse all samples, in triplicate wells. Seed 150 uL/well,
according to template, 96-well plate, 0.5x106 cells/mL
Incubate 24h, add 50 uL/well of resazurin (final conc in wells
55 uM) incubate 2-3h 37°. Read generated fluorescence in
Tecan M200 multiplate reader . Excitation set to 530 and
emission set to 590 nm. Resazurin stock solution 4.4 mM.
The reader is booked for 1hr at 3 occasions on
Tuesday 10/3:
12:00-13:00, 14:00-15:00, 16:00-17:00
530nm 590nm
3. Giemsa/May Grunewald stain.
Consider results from the two viability assays “trypan blue exclusion” and
“resazurin assay.” Which samples from the chemical library affected growth?
Choose 4 samples together with the solvent control and spin out cells on slides
and stain cells with May Grunewald/Giemsa according to instructions given in
Lab 1.
After staining look through the slides and search for a phenotype for that
specific drug.
Estimate ratio “phenotypic cells”/normal cells among 100-150 cells.
Methods to distinguish out stages in the cell cycle:
G0 from G1.
Ki-67 is not expressed in G0 cells
R
G0/G1
S G2/M
Has cells passed the G1 restriction point.
Western blot of cell lysates with Rb-antibodies
Rb
pRb
actin
How to resolve different stages within G2/M
G0/G1
S G2/M
FACS analysis of micro-tubules
Jurkat cells
μ-tubules
Control
Drug
Ref Dipak M et al Nature Comm 2013
Intact cells
Ref: Per Holmfeldt, Umeå university
Perforation of plasma membrane in
presence of saponin and taxol;
Unpolymerised tubulins will diffuse out of
the cell
After fixation incubate cells with
anti-tubulin antibodies followed by
incubation with FITC-labelled
secondary antibodies.
Staining of microtubules for FACS
(copied from “-MG lab methods 2011-“)
Solutions:
MT-STAB
PEM buffer, pH 6.9
0.1 % Saponin (stock 10%)
10 µg/ml RNase (stock 5mg/ml)
50 nM Taxol (stock 1,1mM)
MT-FIX
~50 % PEM buffer, pH 6.9
~50 % PFA in H2O (from 8% stock)
0.1 % Saponin (add 1/100 of a 10 % stock,
which may be stored ~1 week)
BLOCK
90 % PBSA / 10 % FCS / 0,1 % Saponin
WASH
PBSA / 0,1 % Saponin
PI SOL.
PBSA
10µg/ml Propidium Iodide
0,1% Triton X-100 (add 1/100 of a 10 % stock)
10µg/ml RNase
1.
2.
3.
4.
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6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
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18.
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20.
21.
Spin down 500.000 cells per FACS tubes for 2 min, 1100rpm
(250xg).
Aspirate the supernatant and resuspend the pelleted cells
gently using the shaking table.
Extraction of cells; add 400µl 37 °C MT-STAB and incubate at
37 °C for 4 minutes.
Fixation of cells; add 400 µl 37°C MT-FIX and incubate at
37 °C for 12 minutes.
Spin down the cells, 3 min, 1100rpm.
Aspirate the supernatant and resuspend the cells by shaking.
Add 1 ml BLOCK and incubate at 37 °C for 10 minutes.
Spin down the cells, 5 min, 1100rpm.
Aspirate the supernatant and resuspend the cells by shaking.
Add 150 µl primary antibody (anti--tubulin T5168 B-5-1-2
dil 1:250 in BLOCK). NOTE! Include a tube with no addition of
primary antibody to define the background.
Incubate at 37 °C for 60 minutes while shaking.
Add 1 ml WASH.
Spin down the cells, 5 min, 1100rpm.
Aspirate the supernatant and resuspend the cells by shaking.
Add 100 µl secondary antibody (Rabbit-anti-mouse (FITC)
DAKO F0313 dilute 1:20 in WASH).
Incubate at 37 °C for 30 minutes while shaking.
Add 1 ml WASH.
Spin down the cells, 5 min, 1100rpm.
Aspirate the supernatant and resuspend the cells by shaking.
Add 500µl PI SOL and keep samples in refrigerator until
analysis.
micro-tubules är very britle
Aspirate
medium
500 μL
cells
Loosen pellet by
gently shaking
on shakerboard
Extraction of
“free” tubulin
Add 400 μL
MT-STAB 37°
inc 37° 4 min.
Fixation of cells
Add 400 μL MT-FIX
37° inc 37° 12 min
Centrifuge
1100 rpm
3 min
Centrifuge
1100 rpm
2min
Aspirate sup
and shake gently
Blocking of unspecific
protein binding sites.
Add 1ml BLOCK and
inc 10min 37°
Aspirate sup
and shake gently
Add 150 μL Mouse
anti-α–tubulin
Antibody and inc
60min 37° in
shaking
waterbath.
Centrifuge
1100 rpm
5 min
Aspirate sup
and shake gently
Add 100 μL Secondary ab:
FITC Rabbit α mouse IgG,
inc 30min 37° in shaking
waterbath
Add 1 mL
WASH
Centrifuge
1100 rpm
5 min
Add 1 mL
WASH
Centrifuge
1100 rpm
5 min
Aspirate sup
and shake gently
Add 0.5ml PI-soution.
Store cells in fridge
until FACS analysis
Structural effects on microtubules
After FACS analysis of cells stained for u-tubules
Adjust cell cons to approx 0.5x106/mL and resuspend the
cells. Spinn out 0.1x106 cells onto slides. Mount with
“anti-fade” mounting medium and cover with coverslip.
Let dry until next day an inspect the micro-tubule
structure with a fluorescence microscope. Describe the
structrual changes, count the phenotypic cells vs normal
cells.
Microscope slides with wells
an alternative to cytospinning
Adjust cellconc to approx 0.5x106/mL.
Label wells on slide, 12 wells/slide and put it into
”humified chamber”. Pipet 25 μL/well of resuspended
stained cells in PI-sol. Allow cells to sediment onto glass
for 2h in the dark. Remove excess of solution with a small
piece of 3MM, add a drop of “anti-fade” mounting
medium to the well and cover with a round coverslip
11mm diameter.
Safety issues:
• PFA (paraformaldehyde) is skin and
airway irritant and could cause allergic
reactions! Wash exposed skin areas
with water and soap.
• May Grunewald staining solution is
flammable and skin irritant. Rinse
with ethanol followed by water in
large amounts!
• Giemsa is skin irritant. Rinse with
ethanol followed by water in large
amounts!
• PI (propidium iodide) binds to DNA
and should be handle with care. Rinse
with ethanol followed by water in
large amounts!
• All drugs are cytotoxic drugs. Rinse
with ethanol followed by washing of
exposed areas with water and soap.
Group wise presentation:
Fully planned experiments
Reagents and supplies and amount needed as well as a time schedule
Drugs: 200x final conc.
Jurkat cells: Amount of cells and RPMI1640 10% FCS.
Reagents : MT-STAB, MT-FIX, PI-solution, trypan blue solution, resazurin 4.4 mM,
saponin 10%, antibodies, buffers……………………………………………..
Other supplies
Tubes Epp-tubes, multiwell plates, slides, cover slips …………………………………………….
Equipment:
You do not need to include pipett tips , disposable pipets, May Grunewald and Giemsa
staining solutions and mounting media.
Carefully planned time schedule for Monday 10/3 Tuesday 11/3.
Calculations how to obtain original cell conc when cells are diluted 200ul cells + 50 ul
trypanblue.
Format for seeding cells into 96-well plate
for resazurin
Time Slots for group discussions:
Monday 10/3
group
8:00
15, 16, 17
8:30
12,13, 14
9:00
9, 10, 11
12:00
12:30
13:00
6, 7, 8
3, 4, 5
1, 2
C
C
C
C
C
C
S1 S3 S5 S7
S1 S3 S5 S7
S1 S3 S5 S7
S2 S4 S6 B
S2 S4 S6 B
S2 S4 S6 B
C Solvent control
S1 Cells treated with drug #1
S2 Cells treated with drug #2
and so on
B culture medium without cells