Human Histamine H1 Receptor, γ-Irradiated Frozen

TECHNICAL
DATA
SHEET
AequoZen®
Caution: For Laboratory Use. A research reagent for research purposes only
Human Histamine H1 Receptor, -Irradiated Frozen Cells
Product No.: 390-AF
Lot No.: 1002
Material Provided
Cells:
1 x 1 mL frozen aliquot
Format:
~10 x 10 cells/mL in Ham’s F12, 10% FBS with 10 % DMSO
6
Product Information
Cellular Background:
CHO-K1
Parental Frozen Cells (control):
A12 (Cat # ES-000-A12F)
Frozen Cells Info:
Frozen recombinant, irradiated CHO-K1 cells expressing
mitochondrially-targeted Aequorin and the human Histamine H1
receptor.
DNA Sequence:
Identical to coding sequence of GenBank NM_000861.3.
Corresponding Protein Sequence:
Identical to GenBank NP_000852.1.
Storage Conditions:
Store in liquid nitrogen (vapor phase) immediately upon receipt, or
®
maximum 15 days at -80°C. AequoZen is designed for single use
only. Do not refreeze.
Quality Control
®
EC50 for a reference agonist is determined using an AequoScreen assay (Figure 1). Mycoplasma test is performed
®
using MycoAlert Mycoplasma detection kit. We certify that these results meet our quality release criteria.
Histamine dihydrochloride (EC50):
3.3 nM
Mycoplasma:
This cell line tested negative for Mycoplasma.
TDS-ES-390-AF-04
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Recommended Thawing Conditions and Handling of Frozen Cells

Carefully follow instructions below to obtain the expected results. Most Frozen cells are intended to be
assayed immediately upon thawing. Exceptionally, where specified, some frozen cell products require an
overnight incubation in Cell Medium to enable them to perform optimally.

The recommended media catalogue number and supplier reference information are listed in this Product
Technical Data Sheet (last page). Media composition is specifically defined for each cell type and receptor.
The use of incorrect media or component substitutions can lead to altered product performance. Additionally,
the instructions for the preparation of ligands must be carefully followed to avoid ligand precipitation,
degradation or adsorption. Inappropriate preparation may result in a non-representative pharmacology.

The complete thawing procedure must not exceed 30 min. Cell viability below 90% upon thawing may indicate
that the Frozen cells were affected by incorrect thawing procedure and may yield to lower performance.
Ensure the cells are not clumped and are evenly distributed in the assay plates. Gently pipet up and down if
cells are clumped before dispensing the cells. Frozen cells cannot be re-frozen.
®
Assay Medium (for immediate thaw and use):
AequoScreen Assay Buffer (see below)
Cell Medium (for overnight incubation prior to use):
Ham's F-12, 10% FBS
Thawing Cells:

Using appropriate personal protective equipment, rapidly place the frozen aliquot in a 37°C water bath (do not
submerge) until its content is thawed completely. Immediately remove from water bath, spray aliquot with 70%
ethanol and wipe excess. Under aseptic conditions using a sterile pipette, gently resuspend the cells in the
cryovials and transfer content to a sterile centrifuge tube containing 10 mL of the Assay or Cell Medium prewarmed to 37°C, and centrifuge (150 x g, 5 min.). Do not exceed the recommended centrifugal force. Discard
supernatant using a sterile pipette. Gently resuspend cell pellet in 5 mL of appropriate pre-warmed medium by
gently pipetting up and down to break up any clumps. For immediate use, dilute cells to recommended cell
density in Assay Medium.

For an overnight incubation step, plate the cells in Cell Medium in a T75 cm culture flask. Incubate overnight
at 37°C in a humidified atmosphere with 5% CO2. Most cells will adhere but they will not grow, due to the
gamma-irradiation process. To harvest cells, under aseptic conditions, remove media, rinse with 4.5 mL of
calcium and magnesium-free PBS, add 4.5 mL Versene or calcium and magnesium-free PBS/0.5 mM EDTA,
and incubate at room temperature until cells detach (do not exceed 5-10 minutes). Add 9 mL of Assay
Medium, collect the cells, centrifuge (150 x g, 5 min) and resuspend in Assay Medium to the recommended
cell density.
2
Recommended Cell Density per Assay Point (MicroLumat): 25 000 cells/well

Optimal cell density per assay point will depend on the sensitivity of the reader used. The values given here
are the ones determined for using the cells on a MicroLumat Reader, but may need optimization when using
another reader.

Reducing the coelenterazine concentration and loading time in the AequoScreen assay will still result in
some signal, but is expected to result in decreased signal intensity and/or stability, so we do not recommend
reducing these parameters.

Temperature should remain below 25°C during the coelenterazine loading of the cells, and until using the
cells for the readings. Excessive heating by the cell stirrer for example will result in signal loss.
®
TDS-ES-390-AF-04
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Typical Product Data
Histamine diHCl
3.2x10
Emitted Light (RLU)
2.8x10
2.4x10
2x10
1.6x10
1.2x10
Agonist
+6
+6
Histamine
+6
dihydrochloride
EC50
% of Digitonin
(M)
response
2.0 x 10
-8
102
+6
+6
+6
800000
400000
0
-13
-10.5
-8
Log [Ligand], M
-5.5
®
Figure 1: Agonist Response in AequoScreen assay
An agonist dose-response experiment was performed in 96-well format using 25 000 cells/well. Luminescence was
measured with the MicroLumat. Data from a representative experiment are shown. The Z’-factor was calculated for
Histamine dihydrochloride with at least 16 background and 16 maximal signal points (Z’= 0.94).
4x10
3.6x10
Emitted Light (RLU)
3.2x10
2.8x10
2.4x10
2x10
1.6x10
1.2x10
+6
+6
trans-triprolidine
Mepyramine
+6
Antagonist
+6
+6
IC50
(M)
trans-triprolidine hydrochloride
1.8 x 10
-8
Mepyramine
7.5 x 10
-9
+6
+6
+6
800000
400000
0
-13
-10.5
-8
Log [Ligand], M
-5.5
®
Figure 2: Antagonist Response in AequoScreen assay
An antagonist dose-response experiment was performed in 96-well format using 25 000 cells/well. The reference
agonist used was Histamine dihydrochloride, at a final concentration equivalent to the EC80. Luminescence was
measured with the MicroLumat. Data from a representative experiment are shown.
TDS-ES-390-AF-04
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AequoScreen® Assay Procedure (MicroBeta® JET)
Assay Buffer:
DMEM / HAM’s F12 with HEPES, without phenol red (Invitrogen # 11039-021) +
0.1 % protease-free BSA (from 10% solution sterilized by filtration at 0.22 µm).
Store at 4°C.
Coelenterazine h:
To prepare a 500 µM Coelenterazine h stock solution, solubilize 250 µg of
Coelenterazine h (Promega # S2011 or Invitrogen # C6780) in 1227 µL methanol.
Store at -20°C in the dark.
Digitonin:
To prepare a 50 mM Digitonin stock solution, dissolve 1 g of Digitonin (Sigma #
37006) in 16.27 mL of DMSO. Aliquot and store at -20°C.
1.
Cell preparation:
Resuspend thawed cells prepared as exposed above in Assay Buffer at a
5
concentration of 3x10 cells/mL.
2.
Coelenterazine
Loading:
Under sterile conditions, add “Coelenterazine h” at a final concentration of 5 µM
to the cell suspension, mix well. Incubate at room temperature protected from
light and with constant agitation for at least 4 hours (incubation can be extended
overnight).
3.
Cells Dilution:
Dilute cells 3x in assay buffer and incubate as described above for 60 min.
4.
Ligands and plates
preparation:
Prepare serial dilutions of ligands in assay buffer, (2x concentration for agonists,
3x concentration for antagonists). Dispense 50 µL of diluted ligand in a 96-well
Optiplate™. Note: Assay can be miniaturized to 384-well and 1536-well formats.
5.
Agonist Mode
Reading:
Using the reader’s automatic injection system, inject 50 µL of cells (i.e. 5 000
cells) per well and immediately record relative light emission for 20-40 seconds.
Digitonin at a final concentration of 50 µM in assay buffer is used in control wells
to measure the receptor independent cellular calcium response.
6.
Antagonist Mode
Reading:
After 15 minutes of incubation of the cells with the ligand, using the reader’s
automatic injection system, inject 50 µL of the reference agonist at a final
concentration equivalent to the EC80 and immediately record relative light
emission for 20-40 seconds.
7.
Data Analysis:
The luminescent signal is integrated from second 0 to 20-40, and the integrated
value (Area Under the Curve, AUC) is used to draw sigmoidal dose-response
curves.
Important Notes:

Depending on (1) sensitivity of the reader used, (2) plate format used, and (3) assay characteristics wanted,
it is possible to load cells at (a) different concentrations of cells and coelenterazine, (b) with different
subsequent dilution factors, and (c) using different cell numbers per well. This is part of the validation work
when importing an assay to a new reader.

For tips and examples on running AequoScreen assays on different readers, please refer to the
®
AequoScreen Starter Kit Manual available at www.perkinelmer.com/CellLines.
TDS-ES-390-AF-04
Page 4 of 5
®
Materials and Instrumentation
The following tables provide the references of compounds and reagents used or recommended for the
characterization of the human Histamine H1 Frozen cells, as well as some advice on how to use these compounds:
Table 1. References of compounds used for functional characterization
Name
Histamine dihydrochloride
Trans-triprolidine hydrochloride
Mepyramine
Provider
Sigma
Sigma
Tocris
Cat n°
H-7250
T-118
0660
Working Stock Solution
100 mM in water
10 mM in water
10 mM in water
Table 2. References of cell culture media and assay buffers.
Note: The table below lists generic media and additives typically used for PerkinElmer Frozen cells. For product
specific media and additives, please refer to the “Recommended Thawing Conditions and Handling of Frozen Cells”
section.
Name
HAM’s F-12
DMEM
Advanced DMEM/F12 (serotonin receptors)
EMEM
EX-CELL DHFR media (DHFR deficient cell lines)
FBS
FBS dialyzed
Calcium and magnesium-free PBS
DMEM / HAM’s F12 with HEPES, without phenol red
Coelenterazine h
Coelenterazine h
Digitonin
BSA, Protease-free
Trypsin-EDTA
Sodium Pyruvate
L-Glutamine
NEAA (non-essential amino acids)
Provider
Hyclone
Hyclone
Invitrogen
BioWitthaker
Sigma
Wisent
Wisent
GIBCO
Invitrogen
Promega
Invitrogen
Sigma
Sigma
Hyclone
GIBCO
GIBCO
GIBCO
Cat n°
SH30026.02
SH30022.02
12634-010
06-174G
C8862
80150
80950
11010
11039-021
S2011
C6780
37006
A-3059
SH30236.02
11360
25030
11140
Please visit our website: www.perkinelmer.com/CellLines for additional information on materials, microplates and
instrumentation.
This product is not for resale or distribution except by authorized distributors.
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