EnSpire-LFC, 384 well (Cell Assay Microplate, uncoated)

TECHNICAL
DATA SHEET
Microplates
EnSpire-LFC, 384 well (Cell Assay
Microplate, uncoated)
Cellular Label-free Microplate
Part numbers: 6057400, 6057408.
Caution: For Laboratory User. A research product for research purposes only.
Product provided
Microplates:
2 x 384-well microplates per case (6057400)
8 x 384-well microplates per case (6057408)
Format:
Individually packaged
Description
®
The EnSpire -LFC 384-well microplate is an ANSI/SBS standard microplate with a patented optical
®
®
biosensor integrated into each well. It is an integral component of the EnSpire with label-free Corning Epic
technology. The surface of each plate is tissue culture compatible and enables attachment and normal
growth of adherent cells including native cells, recombinant / engineered cell lines and frozen cells, and
primary cells. This label-free enabled microplate can only be used with the EnSpire with label-free
technology and not with other label-free platforms.
Product Information
Handling
Open no more than 1 hour before use. Avoid touching the bottom of
the microplate
Cleaning procedure
Fingerprints can be removed using 100 % Ethanol and a kim wipe.
®
(Kimberly-Clark KIMWIPES Disposable Wipers, part number 2017)
Materials
Cyclic Olefin Copolymer with Glass Bottom
Plate format
384-well
Plate color
Black with clear bottom
Well bottom
Flat
Well bottom color
Clear
Well shape
Round
Well area
0.056cm
Maximum well volume
82 µL
Recommended working volume
15-50 µL
Surface treatment
Tissue culture compatible
Sterile
Aseptically manufactured
Lids included
Yes
Typical seeding density
5,000 – 20,000 cells/well (cell type dependent)
Barcode
Yes (short right hand side)
Regulatory status
Patented Biosensor technology
Shipping conditions
Ambient
Storage conditions
Room temperature
Shelf life
Minimum 3 months from shipment date when stored correctly
2
Quality Control
•
Each well is factory tested for resonant wavelength
•
Tested for growth and morphology of CHO-M1 cells
•
Functionally tested for assay performance
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Applications
•
Assay development, primary and secondary cell-based screening, profiling
•
GPCR pathway identification and validation
•
Endogenous receptor modular
•
Receptor panning
•
Orphan receptor studies
•
Cell proliferation studies
•
ADME/Tox studies
•
Ion channel assays
•
Tyrosine kinase receptors
Cell Lines
•
CHO cells
•
HeLa cells
•
Cos 7 cells
•
A431 cells
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Specifications
Microplate Dimensions
Number of rows
16
Number of columns
24
Height
14.22mm without lid (15.50mm
with lid)
Depth of well
10.92 mm
Well diameter
3.40mm at top, 2.82mm at bottom
Well volume
82 µL
Top left corner
X = 12.13, Y = 8.99
Top right corner
X = 115.63, Y = 8.99
Bottom left corner
X = 12.13, Y = 76.49
Bottom right corner
X = 115.63, Y = 76.49
Bottom clearance
2.5 mm
Microplate flatness (global
flatness)
50 µm
Microplate flatness (within well
flatness)
0.3 µm
Thickness of glass bottom
0.8 mm
Biosensor position
True position of 250 µm in any
direction from SBS center points
Table 1. Microplate specifications.
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Figure 1. Microplate dimensions and cross section of well
Standard Cell Based Assay with PerkinElmer Human Adrenergic β2 Receptor γ–
irradiated Frozen Cells using EnSpire-LFC, 384-well plate (Cell assay microplate,
uncoated)
This protocol describes how to perform an EnSpire label-free cell-based assay that consists of antagonist
and agonist additions in an EnSpire-LFC, uncoated 384-well plate.
Note: You must use regular Fetal Bovine Serum (not heat inactivated).
Materials
2+
2+
•
HBSS with Ca
•
1M Hepes, pH 7.4 (Invitrogen cat# 15630-080)
•
F12K Nutrient Mixture (Gibco cat# 21127)
•
Fetal Bovine Serum (Gibco cat# 10099) (not heat activated)
•
OPTIONAL PenStrep (Gibco cat# 15070)
•
Propranolol (Sigma cat# P8688)
•
Isoproterenol (Sigma cat# I6504)
•
1 vial Human Adrenergic β2 Receptor γ-Irradiated Frozen Cells (PerkinElmer cat# ES-034-CF)
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and Mg
(Invitrogen cat# 14025-126)
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•
DMSO (Sigma cat# D8418)
•
EnSpire-LFC, 384-well (Cell assay microplate, uncoated) (PerkinElmer cat# 6057400)
•
Tissue Culture Incubator (37°C, 5% CO2 or as appropriate for specific cell type)
•
Water bath, 37°C
•
Cell counter
•
Microscope
•
384-well multichannel pipettor (preferably 16-channel)
•
Aspiration Wand (VP Scientific cat# VP186LP), including vacuum pump source
•
Standard Laboratory Grade Tape used to tape ends of Aspiration want so that it leaves 10 µL
residual volume in test plate (tape and test wand prior to assay)***
•
384-well polypropylene microplate (PerkinElmer cat# 6008590)
•
15 mL and 50 mL conical tubes
•
Manual pipets (10 µL - 1000 µL)
•
Multichannel pipets, 384 and 96-well plate compatible (5 – 50 µL at least manual)
•
Eppendorf tubes (1.5 mL)
•
Foil plate seals (VWR cat# BK538619)
•
18 MΩ distilled, filtered water (or cell culture-grade)
•
0.22 µm cellulose acetate filter system (1L capacity)
•
Cell culture hood equipped with vacuum pump source
•
Bleach
•
Ethanol (70% in spray bottle)
•
Reagent reservoirs (100 mL)
•
Glass or plastic storage bottles for making buffers (250 – 1000 mL)
Reagents Preparation (Day 1)
1. Medium preparation in cell culture hood (complete media).
o 500 mL F12K Nutrient Mixture
o 50 mL Fetal Bovine Serum (not heat inactivated)
o 5 mL Penicillin-Streptomycin (OPTIONAL)
o Filter using 0.22 µm cellulose acetate filter system
o Store at 4°C
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2. Assay buffer #1 preparation – make on Day 2 (HBSS/20mM Hepes, pH 7.4)
o 1 liter Hank’s Balanced Salt Solution
o 20mL of 1M HEPES stock
o Store at room temperature
3. Assay buffer #2 preparation – make on Day 2 (HBSS/20mM Hepes/1% DMSO)
o 300 mL assay buffer #1 (HBSS/20 mM Hepes)
o 3 mL DMSO
o Store at room temperature
4. Antagonist (Propranolol) Preparation (100 mM)
o Weigh 25 mg of Propranolol in 1.5 mL Eppendorf tube
o Add 1 mL DMSO, vortex
o Store aliquots at -20°C
5. Agonist (Isoproterenol) Preparation (100 mM)
o Weigh 25 mg of Propranolol in 1.5 mL Eppendorf tube
o Add 1 mL water, vortex
o Store aliquots at -20°C
Cell Seeding (Day 1)
1. Warm complete media to 37°C in water bath.
2. Put on gloves and spray down cell culture hood with 70% ethanol (be sure that all supplies brought into
the hood are sterile or sprayed with 70% ethanol).
3. Take EnSpire-LFC, 384-well uncoated “test plate” out of package and place in hood.
4. Using multichannel pipettor, add 10 µL complete media to columns 1-12 of test plate.
5. Centrifuge plate at 800 RPM for 1 minute and place back in hood.
6. Remove vial of β2AR cells from liquid nitrogen (wear gloves and eye wear).
7. Hold vial of β2AR cells in 37°C water bath (do not submerge) until vial is 75% thawed.
8. Spray vial with 70% ethanol and put in cell culture hood.
9. Add 1 mL of complete media slowly to vial and aspirate up and down 2 times.
10. Transfer all contents of vial to a 50 mL conical tube containing 8 mL complete media (total volume 10
mL).
11. Centrifuge 50 mL tube of cells at 1500 RPM for 5 minutes and remove supernatant (to wash cells).
12. Re-suspend pellet in 5 mL complete media and count cells.
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13. 500 cells / µL (15,000 cells/well/30 µL), or 500,000 cells/mL, 7 mL of cells is sufficient for half of a 384well plate, so roughly 3.5 million cells are required to plate at a density of 15,000 cells/well in half of a
384-well plate.
14. Dilute cells appropriately (from 5 mL suspension) in complete media and pour into sterile reservoir.
15. Add 30 µL of cells to columns 1-12 of test plate using multichannel pipettor (be careful not to touch
bottom of test plate, but also be careful not to pipet too high to create air bubbles).
16. Incubate lidded plate in hood for 30 minutes to stabilize.
17. Put test plate in 37°C incubator overnight.
Buffer Exchange (Day 2)
1. Look at cells in plate under microscope (note degree of confluency).
2. Prepare aspiration wand prior to assay by taping ends so that residual volume left in plate is 10 µL ***
3. Media/Buffer exchange using aspiration wand (assay buffer #2 HBSS/20mM Hepes/1% DMSO)
o Remove 30 µL media from assay plate using aspiration wand for net 10 µL
o Add 25 µL assay buffer
o Remove 25 µL from assay plate for net 10 µL
o Repeat above add and remove steps three times for a total of four 25 µL exchanges
o Add 20 µL assay buffer for net 30 µL
4. Let cell plate equilibrate for 2 hours near EnSpire (to keep temperature constant) – do not return plate to
tissue culture incubator.
5. While cell plate is equilibrating, prepare compound dilution plates.
Compound Source Plate Preparation (Day 2)
1. Two 384-well polypropylene microplates will be needed, label plates “add 1-Antagonist” and “add 2Agonist”.
2. Take out frozen aliquots of propranolol and isoproterenol.
3. Buffers #1 (HBSS/20 mM Hepes) and #2 (HBSS/20 mM Hepes/ 1% DMSO) will be needed.
4. Using a multichannel pipettor add 60 µL buffer #2 (with DMSO) to columns 1-6, rows B-P only for
columns 4-6 (see plate map #1 below of “add 1-antagonist” plate).
5. Dilute 100mM propranolol to 1 mM (1:100 by adding 2 µL 100 mM propranolol to 198 µL Buffer #1 (no
DMSO).
6. Further dilute the propranolol to 4 µM working concentration (1 µM final concentration), dilute 1 mM
propranolol (1:250 by adding 4 µL 1 mM propranolol to 996 µL buffer #2).
7. Add 80 µL 4 µM propranolol to wells A4-A6 of “add 1-antagonist” plate.
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8. Perform a 1:4 serial dilution of propranolol in columns 4-6 starting at Row B (20 µL stock from Row A into
60 µL buffer #2) directly into 384-well round bottom polypropylene microplate through row N.
9. Cover “add 1-antagonist” plate and set near EnSpire.
10. To “add 2-agonist”, add 60 µL buffer #2 (with DMSO) to columns 1-3, rows B-P only (see plate map #2
below of “add 2-agonist plate), and to columns 4-6, rows O-P only.
11. Dilute 100 mM isoproterenol to 1 mM (1:100 by adding 2 µL isoproterenol to 198 µL buffer #2).
12. Further dilute the compound to 5 µM working concentration (1 µM final concentration), (1:200 by adding
5 µL propranolol to 995 µL buffer #2).
13. Add 80 µL 5 µM isoproterenol to wells A1-A3.
14. Perform a serial dilution to isoproterenol in columns 1-3 starting at row B (20 µL stock from tow A into 60
µL buffer #2) directly into 384-well round bottom polypropylene microplate through row N by diluting 5
µM stock to 5 nM (1:1000 by adding 5 µL of 5 µM stock to 4995 µL buffer #2).
15. Add 60 µL of 5 nM isoproterenol to columns 4-6, rows A-N.
16. Cover “add 2-agonist” plate and set near EnSpire.
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Add 1- Antagonist Compound Source Plate (4x)
1
2
3
4
4 µM
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4 µM
4 µM
1:4 dilutions
60 µL Buffer #2
4 µM stock propranolol
60 µL buffer #2 where dilution series is performed
no compound wells
Add 2- Agonist Compound Source Plate (5x)
1
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
5 µM
2
3
4
5
6
5 µM
5 µM
1:4 dilutions
5nM isoproterenol
(fixed dose)
5 µM stock isoproterenol
60 µL Buffer #2
60 µL buffer #2 where dilution series is performed
5 nM isoproterenol (fixed dose)
no compound wells
Figure 2. Antagonist and Agonist compound source plate maps.
Assay “Baseline Measurement” (Day 2)
1. Open EnSpire Manager Software.
2. Create a New Protocol (label free, 384-well, cell-based).
3. Add a “Measurement A”, add 4 repeats for the baseline (~5 min.), and add 60 repeats for the final read
(~60 min.).
4. Name assay as appropriate (i.e. β2AR CBA date, initials).
5. Save Assay.
6. Load Plate and start baseline reads. A slight negative drift in pm is normal.
7. Once baseline read is complete, plate will automatically unload, and Assay can be performed.
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Assay “Step 1 – Add 1” (Day 2)
1. Working quickly, use a multichannel pipettor to transfer 10 µL of contents of “add 2-agonist” compound
source plate to the corresponding wells of test plate, mix carefully by pipetting 1X, and be careful not to
touch the bottom of the wells of the test plate.
2. Quickly reload plate into EnSpire for final read #1 (~60 min.).
3. Plate unloads automatically.
Assay “Step 1 – Add 2” (Day 2)
1. Working quickly, use a multichannel pipettor to transfer 10 µL of contents of “add 2-agonist” compound
source plate to the corresponding wells of test plate, mix carefully by pipetting 1X, and be careful not to
touch the bottom of the wells of the test plate.
2. Quickly reload plate into EnSpire for final read #2 (~60 min.).
3. Analyze data using PKI Workstation Software and calculate respective EC50/IC50 values.
4. Calculate respective EC50 values.
Good Lab Practice
1. When preparing test plates, always make all solutions first and open the plate package last to minimize
exposure to air.
2. Always use the same source of reagents and buffers with an assay as well as when running multiple
assays on a given day.
3. When using the hand pipettors, be sure that bubbles are not being pulled up in the tips. Also try to watch
the fluid level in the tips as it is being dispensed to ensure the same volume goes into each well.
4. Avoid fluctuations in temperature wherever possible by keeping buffers, compound plates, etc. in the
same room.
Working with a few wells at a time
It is possible to work with a few wells at a time. Open the plate in a hood if only a few wells are to be used.
Keep the lid on whenever transferring the plate. Each time the plate lid is removed, you are increasing the
risk of contamination of your cells.
Please visit our website for more information: www.perkinelmer.com/label-free
This product is not for resale or distribution except by authorized distributors.
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PerkinElmer, Inc.
940 Winter Street
Waltham, MA 02451 USA
P: (800) 762-4000 or
(+1) 203-925-4602
www.perkinelmer.com
For a complete listing of our global offices, visit www.perkinelmer.com/ContactUs
Copyright ©2011, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. Corning and Epic are registered trademarks of Corning Incorporated. All
other trademarks are the property of their respective owners.
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