This document provides information about setting up and conducting experiments with isolated organs and tissue rings, including: 1. Describing the mechanical setup for a four-channel system bath for isolated organs. 2. Explaining the preparation of Krebs-Hanseleit solution and common drugs used. 3. Outlining typical experiment protocols, including stabilizing tissues, pre-contraction testing, and assessing endothelial function. 4. Noting that each experiment begins by preparing Krebs-Hanseleit solution and activating the system before surgery and setting rings in wells.

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Table of Contents
1. Mechanical settings setup
1
2. Preparation Krebs - Hansenleitove solution 2
3. Preparation of drugs 3
4. protocols 4
5. Data processing 5
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2. MECHANICAL SETUP FEATURES
1.1. Parts of four-channel standard system bath for isolated organs
 A four-channel system standard bath for isolated organs (tissue rings and strips) with its terminal
equipment (pipes, cables, etc.)
 Four-enhancer measured force
 Isometric force converter
 Of software for the data collection and processing
 The system for receiving and transmitting siganala with system baths for isolated organs in the
computer (four-channel with the option to upgrade to osamkanalni system)
 The heater and the circulation water tank 20 allowing the adjustment of the temperature in the range
20-50 ° C and with a precision of ± 0.1 ° C

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2. PREPARE KREBS-HENSELEIT'S SOLUTION
Prepare solution of ingredients according to Table 2.1. First, the Erlenmeyer flask pour in 2 l of
deionized water and put the proper MAGNETIC set and all the magnetic strier. Gradually add all
ingredients and solution mješajjte on magnetic striere 10 minutes until all ingredients are dissolved.
Ingredient Focus / mM Weight / g Weight / g
deionized water - 2 L 4 L
NaCl 113 13.79 27,580
KCI 4.7 0,701 1,402
CaCl2 x 2H2O 2.5 0.735 1,470
EDTA 0,026 .0152 .0304
MgSO4 x 6H2O 1.2 .593 1,185
KH2PO4 1.2 0,327 .653
NaHCO3 22 3.698 7.395
Glucose - 3,998 7,999
Table 2.1 - Ingredients Krebs - Henseleit solution

4. 3. PREPARATION OF LIQUIDS
The most commonly used drugs in experiments with an isolated blood vessels are shown in Table 3.1.
When preparing the drugs it is important to note that the drug (maybe more appropriate name would be active
substance) when the application in the basin further diluted.
The dilution factor (FR) derived from the ratio of the volume of wells (UK) and the volume of drug
solution administered to the well of (VA). Drug concentration in a solution which is prepared (cP) is
calculated so that the drug concentration in the well want to achieve (cB) multiplied by the dilution factor
(FR).
Wells volume (Vb) of our setup is 10 mL, so if we apply the drug solution 100 ml of this is diluted
when it has 100 times (FR 10 = 000μl / 100μl). It follows that the concentration of the drug solution (cP)
which is prepared to be 100 times higher than the concentrations that we want to achieve in the well
(CP = 100 x BW).
The drug / active
substance
MA (g / L)
The
concentration
of the
preparation of
a medicament
The concentration
of medicament in the
well of undiluted
Concentration in
the assay
KCI 74.56 3 M 60 mM (200μL) 60 mM
noradrenaline 205.0 10 mM 10-4
Guinea Pig:100 x (10-
6)
Rat: 1000x (10-7)
acetylcholine 181.7 10 mM 10-4
Guinea Pig:10 x (10-
5)
Rat: 100 x (10-6)
L-NAME 268.0 20 mM 3x10-4 M 3x10-4 M
indomethacin 358.0 10 mM 10-4 10x (10-5 M)
clotrimazole 344.8 10 mM 10-4 10x (10-5 M)
Table 3.1 - the most used drugs and concentration

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FR = VB / VA
UK - volume pools
VA - volume of the drug solution is
applied to the well of
cP = FR x cB
cB - drug concentration in the well cP
- concentration of drug to prepare
3.1 mmost commonly used formulas
The wells [Ml]
FR =
VAinfarctions [Ml]
cP =
FR x cB
n [mole]
substances
F =
VP[L]
nsubstances = Mrtvari [G / mol]
msubstances [Mr]
Mmatter
cP = Mr.matterand
VP
=
Mtwara
TheP Mrtvari

6. VB = 10 ml = FIXNO, certain setup, VA
= 100 l = determine alone, 100 uL
practical
VP = 10 ml = Determine themselves
better to prepare a larger volume of
solution, weighed and 1 ml per store at -
20 ⁰C
Mtwara[Mr] = cP [Mol / L] x Mrtvari[G / mol] x VP[L]
3.2 Preparing drugs for a trial
potassium chloride
Activity Causes a change of the action potential in the smooth muscle
cells of blood vessels and contraction
Using The most commonly used to check the vitality of the blood
vessels at the beginning of the protocol
It can be used for prekontrakciju blood vessels
Concentration of the solution which
is prepared
3M
ingredients solution 11,184 g of KCl in 50 mL of deionized water
Storage Stored at + 4 ⁰C
TB - volume pools
TA - volume of solution that is applied
TP - otopineljeka volume to prepare
cB - drug concentration in the well
cP - drug concentration by preparing
n - amount of drug
Mr.- molecular weight drug
m - weight of the

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The concentration in the well if the
if 200 ml of solution administered in
prpremljene well of
60 mM
The desired concentration of the
Protocol
60 mM
additional dilution Not
The volume which is administered 200 uL
VB = 10 ml
VA (KCl) = 200 uL Fr = 10 ml / 0.2 ml = 50 x
VP (KCl) = 0.05 = 50 ml L
CB (KCl) = 60 mmol / L = 0.06 mol / L
CP (KCl) = 50 x 0.06 = 3 mol /
L MW = 74.56 g / mol
m (KCl) = 3 mol / L x 74.56 g / mole x 0.05 L
= 11.184 g
Noradrenalin
Activity It works directly on the GMS blood vessels causing their
contraction. Acting via α- and β- adrenergic receptors coupled
with G protein
Using For initial prekontrakciju

8. Concentration of the solution which is
prepared
10 mM
ingredients solution 20.5 mg in 10 ml of deionized water
Storage Storage -20 ⁰C in Eppendorf tubes at 1 ml
The concentration in the well if the
if 100 ml of solution administered
in prpremljene well of
10-4 M
The desired concentration of the
Protocol
10-7 M
additional dilution To 1000x (3 times successively 100 l of drug solution in 900 ml
deionized water)
The volume which is administered 100 uL
VB = 10 ml
VA (ON) = 100 uL Fr = 10 mL / 0.1 mL = 100 x
VP (ON) =10 mL = 0.01 L
CB (KCl) = 10-4 mol / L
CP (KCl) = 100 x 10-4 = 10-2 mol / L MW = 205 g / mol
m (NA) = 0.01 mol / L x 205 g / mole x 0.01 L
= 0.0205g = 20.5 mg
acetylcholine
Activity Muscarinic receptors on the surface of endothelial cells and
stimulates their NO synthase (eNOS) in the synthesis of NO
from L-arginine. NO diffuses through the GMS where cGMP-
A promotes relaxation and the ultimate result GMS
vasodilation
Using To test the intactness and endothelial function
Concentration of the solution which
is prepared
10 mM
ingredients solution 18.2 mg in 10 ml of deionized water

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Storage Storage -20 ⁰C in Eppendorf tubes at 1 ml
The concentration in the well if the
if 100 ml of solution administered
in prpremljene well of
10-4 M
The desired concentration of the
Protocol
To check the intactness of endothelium: 10-6 M
To check the function of the endothelium: 10-9 - 10-5 M
additional dilution To check the intactness of endothelium: YES 100x (2 times
successively 100 ml solution of the drug in 900 ml of
deionized water) The functional endothelium: YES 100x (5
times in succession
100 ml solution of the drug in 900 ml deionized water)
The volume which is administered To check the intactness endothelial 100 uL 10-6 M
The functional endothelium: 10-9 100 uL, 90 uL 10-8 - 10 -5
M
VVBA (= ON) = 10 ml 100 uL Fr = 10 mL / 0.1 mL = 100 x
VP (ON) = 10 mL = 0.01 L
CCBP ((KCl) KCl) == x 10100-4 mol / L 10-4 = 10-2 mol / L
Mr = 181,7 g / mol
m (NA) = 0.01 mol / L × 181.7 g / mole x 0.01
L
= 0.0182
g
= 18.2 mg

10. L-NAME
Activity L is -Name blocker endothelial NO - synthase (eNOS), and
consequently an inhibitor of ACh-mediated relaxations
Using To test the mechanisms responsible for this changed endothelial
function due to certain pathological conditions
Concentration of the solution which is
prepared
20 mM
ingredients solution 0.0134 g in 2.5 ml of deionized water
Storage Always prepare fresh solution for test
The concentration in the well if the if
150 ml of solution administered in
prpremljene well of
3x10-4 M
The desired concentration of the Protocol 3x10-4 M
additional dilution Not
The volume which is administered 150 uL
Indometacin
Activity Nonsteroidal anti-inflammatory drug which inhibits the
synthesis of prostaglandins.
Using To test the mechanisms responsible for this changed endothelial
function due to certain pathological conditions
Concentration of the solution which is
prepared
10 mM
ingredients solution 0.0179 g in 5 mL of deionized water
Storage Always prepare fresh solution for test
The concentration in the well if the
if 100 ml of solution administered
in prpremljene well of
10-4 M
The desired concentration of the
Protocol
10-5 M
additional dilution To 10x (1 time 100 ml of solution of the drug in 900 ml deionized
water)
m (NA) = 0.02 mol / L x 268.0 g / mol x 0.005 L
= .0182 M
= 18.2 mM

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The volume which is administered 100 uL
Klotrimasol
Activity A specific inhibitor of CYP450 (CYP450 oxidase) in the metabolism
of arachidonic acid.
Using To test the mechanisms responsible for this changed endothelial
function due to certain pathological conditions
Concentration of the solution which is
prepared
10 mM
ingredients solution 0.01724 g in 5 mL of deionized water
Storage Always prepare fresh solution for test
The concentration in the well if the if
100 ml of solution administered in
prpremljene well of
10-4 M
The desired concentration of the Protocol 10-5 M
additional dilution To 10x (1 time 100 ml of solution of the drug in 900 ml deionized
water)
The volume which is administered 100 uL

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The fourth experiment and protocols
Each experiment begins preparing Krebs - Henseleit solution and activation system. Activating includes starting
of the pump system which heats (set at 37 ⁰C), launch amplifier, oxygen and open the computer and SpeI Isosys
Advanced software. Then you can access the surgery. Surgery should be fast and while still seeking to remove as much fat
and connective tissue. The protocol enter setting rings to the wells.
The protocol consists of several components: stabilizing blood vessels, pre-contract initial test, and testing the
intactness of endothelial function and the specific test (inhibition of specific enzymes, function test of smooth muscle cells
and the like.).

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16. POKUSINAIZOLIRANIMAOR TNIMPRSTENOVIMA
5th Data Processing
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