PT950 myh 是什么意思_百度知道
PT950 myh 是什么意思
请问戒指上有ＰＴ９５０ＭＹＨ是什么意思啊
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是钯金95%的含量~！ myh是品牌名称~！
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出门在外也不愁1.6mpa用在水管上是什么意思?_百度作业帮
1.6mpa用在水管上是什么意思?
1.6mpa用在水管上是什么意思?
S4 管材规格（与壁厚有关）1.6MPA 管材设计承载压力C=1.25 管道总体使用系数C(即安全系数)32*3.6 管材外径及管材壁厚
可以承受的最大压强
1MPa＝9.8公斤 1.6MPaX9.8＝15.68（公斤）这是公称压力的单位就是能承受的压力为16公斤
最大可承受1.6×10^6帕斯卡的压强营养的意思是什么?还有柔软 柳絮 喧闹 梳妆 虎牙_百度作业帮
营养的意思是什么?还有柔软 柳絮 喧闹 梳妆 虎牙
还有柔软 柳絮 喧闹 梳妆 虎牙
营养 yíngyǎng[nourishment] 动物或植物摄取和利用食物过程的总和,在动物,则典型地包括摄食、消化、吸收和同化营养 yíngyǎng[nourish] 有机体从外界吸取养料来维持生命营养身体营养意义：指某些东西比较对人或者事物有用.如：你的话很有营养,
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锡哨Irish Whistle),它还被叫做便士哨(penny Whistle),锡口哨(Tin Whistle),它是锡哨笛类的一种，长度较短，音调较高，多为银白色，黑色，黄铜色·.外文名Irish Whistle颜&&&&色多为银白色，黑色，黄铜色·.
爱尔兰锡哨哨笛起源于的箫，11世纪传入。15世纪爱尔兰宫廷就有关于御用哨笛手的记载，哨笛有六个孔，俗称六孔箫笛，或者6孔哨笛。19世纪前页，一个叫Clarke的人在开办了一个哨笛工厂，这是第一次哨笛以作坊生产的方式出现。哨笛一般由金属制作，也有木制的，锡皮打制的哨笛称为锡哨笛，吹气口为塑料或者木片制成，这种哨笛在爱尔兰传统音乐里使用最为广泛，所以又被称作为爱尔兰哨笛.在上，曾经有许多街头艺人为1便士用哨笛表演一首曲子，所以哨笛也被称为便士哨笛（penny Whistle)这种乐器音色柔美,大家应该都听过它,席琳狄昂为电影《泰坦尼克》演唱的主题歌&我心依旧&(My heart will go on） 主要就是用这种乐器伴奏的.爱尔兰画眉(Song of the Irish Whistle)也主要是使用这种乐器演奏.
新手上路我有疑问投诉建议参考资料 查看Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering(实验步骤)
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[Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering(实验步骤)] 实验材料
PBS [关键词:实验步骤]…
Sodium phosphate
Potassium phosphate
Fine Science
Fine Science
Fine Science
micro-scissors
Fine Science
Fine Science
Scientific
Absorbent bench
Scientific
Scientific
Scientific
sterile transfer
Scientific
collagenase
Worthington
Teflon rod 1/4 inch
McMaster-Carr
Teflon tube 1/4 inch ID, 1/2
McMaster-Carr
Silicone O-Ring 1/4 inch ID,
1/2 inch OD
McMaster-Carr
Teflon tube 1/4 inch ID,
5/16 inch OD
McMaster-Carr
Kendall monoject syringes
Scientific
BD syringe
Scientific
Fibrinogen
Invitrogen
0.2 micron
Scientific
40 micron cell
Scientific
22-363-547
0.45 micron bottle top
pre-filter
18G 1 1/2 inch long
Scientific
21G 1 inch
Scientific
Scientific
Penicillin-streptomycin
Invitrogen
horse serum
Fetal bovine
Invitrogen
aminocaproic
Paraformaldehyde,
Electron Microscopy
Optimal cutting temperature
2-methylbutane
Mouse MYH1/2/4/6 primary
Santa Cruz
Biotechnology
Rabbit Connexin 43 primary
Cell Signaling
Technology
Dylight 549-conjugated
donkey anti-mouse secondary antibody
Jackson ImmunoResearch
Laboratories
715-505-151
Dylight 488-conjugated Donke
anti-rabbit secondary antibody
Jackson ImmunoResearch
Laboratories
711-485-152
Invitrogen
1. Neonatal cardiomyocyte isolation -
preparation (day before)
Solutions created in this section:
PBS-Glucose solution, stop media.
Prepare a PBS-glucose
solution by adding 5 mL penicillin-streptomycin (100 units/ml and
100 &g/ml respectively) and 1.98 g of glucose to 250 ml 1x
sterile phosphate buffered saline (PBS) and bring solution volume
to 500 ml with additional sterile 1x PBS.
Prepare stop media by adding
25 ml FBS and 5 ml of penicillin-streptomycin (same concentration
as above) to 250ml sterile Dulbecco"s Modified Eagle"s Medium
(DMEM) and bring the volume to 500 ml with sterile DMEM before
sterile filtering through a 0.2 micron filter.
Sterilize surgical
instruments needed for isolation by autoclaving: a hemostat, #5
forceps, large scissors, micro-scissors, and a scalpel handle (#4).
2. Neonatal cardiomyocyte isolation -
preparation (day of harvest)
Be sure to maintain sterility
Solutions used in this section:
PBS-glucose solution, Betadine
For each litter, take the two
sterile 100 mm petri dishes, place them in the hood and fill with
?10 mL of ice-cold PBS-glucose. These should then be placed in an
ice bucket filled with ice in the sterile culture hood.
Place a 250 mL beaker with
30-40 mL of Betadine into the hood.
Add 50 mL/litter of
PBS-glucose into a bottle, seal and place into a 37°C water bath.
For each person, place an
absorbent bench underpad on the hood work surface and place a
sterile drape on top being careful not to touch the center work
area of the sterile drape. Dump the surgical instruments and a 4 x
4 gauze pad onto the sterile drape without touching the
instruments. Open a sterile #20 scalpel blade and dump onto drape,
again being careful not to touch with non-sterile gloves.
Take the pups from the dam
and place into opaque container, place pups into the hood
Put on sterile gloves
Fold the gauze into fourths,
clamp with hemostat and place into Betadine beaker.
Put the scalpel blade onto
the scalpel handle and set aside.
3. Neonatal cardiomyocyte isolation -
heart dissection
Solutions used in this section:
Betadine, PBS-glucose solution
Pick up the pup in your non-
dominant hand by pinching skin between shoulder blades between the
thumb and index finger. Using the large scissors, decapitate the
pup in one cut. Be sure to cut from the back of the pup forwards,
to ensure that the spine is completely severed.
Swab the pup"s chest with the
betadine soaked gauze. Secure the pup by pinching the shoulder
blades together. Perform partial thoracotomy to expose the heart.
Increase applied pressure, thereby forcing the heart past the ribs
for scalpular dissection.
Run the scalpel blade behind
the heart to sever the great vessels and remove the heart. Place
the heart in the petri dish containing PBS-glucose that is on ice.
Repeat steps 1-3 for each pup
in the litter.
4. Neonatal cardiomyocyte isolation -
myocyte isolation
Solutions created/used in this
section: PBS-glucose solution, collagenase solution, stop solution
Once the hearts have been
isolated, remove any residual blood and connective tissue by
rinsing in ice-cold PBS glucose solution, remove the top 1/3 of
the heart to isolate only the ventricular tissue and place into a
fresh petri dish with ice-cold PBS glucose, prepared earlier.
Carefully mince the hearts
into ?1 cubic mm using the micro-scissors and the forceps.
Take a sterile transfer
pipette, cut off the tip using scissors so that the mouth of the
pipette is ?3 mm in diameter. Use the pipette to transfer the
tissue pieces and all of the solution into a 50 ml conical and
place on ice.
Weigh out 15,000 units per
litter of pups of type II collagenase (units/mg is dependent on
the lot) and place into the bottle of 37°C warmed PBS-glucose
prepared previously to create collagenase solution. Mix well and
sterile filter into a separate bottle. Place back into the 37°C
water bath. Place the stop solution into the 37° C water bath as well.
Allow the minced tissue to
settle to the bottom of the centrifuge tube. Remove the
supernatant until the total volume is ?10 mL. Add 7 ml of
collagenase solution to the centrifuge tube.
Put the conical tube with the
tissue pieces and the collagenase into a tube rack on an orbital
shaker inside a 37° C incubator or oven. Turn the orbital shaker
on at approximately 60 rpm and close the door. Set a timer for 7
minutes. Be sure to place the collagenase back into the water bath
to keep it warm.
When the timer goes off,
bring the conical back into the hood. Also bring the warm
collagenase and stop solution into the hood. Gently titrate the
tissue pieces 5-7 times to break them up. After titration, allow
the pieces to settle to the bottom (2-3 minutes). Aspirate off as
much of the supernatant as possible being very careful not to suck
up the tissue pieces. Afterwards, add 7 ml of collagenase solution
to the tissue pieces and place back into the incubator on the
shaker for 7 minutes.
For each remaining step,
gently titrate 10 times to break up the tissue pieces. Once the
tissue pieces settle, draw the supernatant off and collect it in a
separate 50 ml conical. Add 7 ml of collagenase to the tissue
pieces and digest again for 7 minutes. To the supernatant tube,
add 10 mL of stop solution with a different serological pipette
after each addition of supernatant from the digestion.
Repeat until all of the
collagenase has been used (7 steps in total).
10)After the final digestion step,
take the conical with the cell solution and filter through
70&m cell sieve into fresh conical.
11)Spin cells down at 100g for 5
minutes and resuspend in 20 mL of DMEM to be counted using a
hemocytometer, and place the cells on ice.
12)Place 50 &l of the cells
into a Trypan Blue solution (75 &l Trypan Blue, 125
&l PBS), mix well before placing 10 &l in the
hemocytometer for counting. Live cells are clear while dead cells
are blue. Expect approximately 3 million cells per rat pup, with a
viability of approximately 80-90%.
5. Casting fibrin gel constructs -
preparation for creating fibrin gels (done well in advance)
Solutions created in this section:
fibrinogen stock solution, thrombin stock solution, Pluronics
solution, myocardial construct media.
Prepare a 33 mg/mL stock
solution of fibrinogen in 20 mM
buffer in 0.9% saline by
slowly mixing fibrinogen into the HEPES buffered saline over
several hours at 37°C. Allow the solution to settle overnight at
2-8°C. Warm the solution to 37°C. The solution is sterile filtered
through a series of consecutive filters: 40 &m cell
strainers, 0.45 &m bottle top filters with glass
pre-filters, and 0.2 &m bottle top filters with glass
pre-filters. The solution is aliquoted into 1 mL and 3 mL aliquots
and stored at -20°C.
Prepare a 25 U/mL stock
solution of thrombin by adding 500 U of thrombin to 18 mL of 0.9%
saline and 2 mL of sterile deionized water, sterile filter through
a 0.2 &m filter, aliquot into 500 &l and 250
&l aliquots and freeze at -80°C.
Prepare a 5% w/v Pluronics
F-127 solution, by dissolving 50 g of Pluronics F-127 to 700 mL of
deionized water. Bring solution volume up to 1L with additional
deionized water. Sterile filter with 0.2 &m filter. The
Pluronics solution can be used up to three times before replacing
if sterile filtered after each use.
Prepare myocardial construct
media by adding 10% horse serum, 2% fetal bovine serum, 1%
penicillin-streptomycin, and 6 mg/mL ?-aminocaproic acid into
DMEM. 50 &g/ml of ascorbic acid and 2 &g/ml of
insulin in 25 &M HEPES need to be added immediately before feeding.
Assemble mandrel by putting
together one teflon rod, one teflon sleeve, two teflon washers
with a notch removed for injection purposes, and two rubber
O-rings (see Figure 2a). Autoclave before use.
Take 6cc syringe casings for
the outer part of the mold and 3cc syringe casings to be used as
plungers, and prepare them by cutting off the luer -lock ends and
autoclaving (see Figure 2a).
6. Casting fibrin gel constructs -
preparation for creating fibrin gels (right before making
the fibrin gel constructs)
Solutions used in this section:
Pluronics solution
Sterile filter the 5%
Pluronics F-127 solution with a 0.2 &m filter before use.
Place mandrels and syringe casings into 5% Pluronics solution in a
1 L beaker in the hood. Leave the parts soaking in the Pluronics
solution for 2-3 hours in the hood to ensure complete coating. The
Pluronics solution coats the mandrels and prevents the fibrin gel
from adhering to the mandrels.
After the 2-3 hour
incubation, pour the 5% Pluronics solution back into the bottle,
place sterile drapes down on the surface of the hood and wear
sterile gloves to construct the molds.
Place the constructed
mandrels into the 6 cc syringe casings, using the 3cc syringe as a
plunger to ensure a tight seal between the o-rings and Teflon washers.
7. Casting fibrin gel constructs via
injection molding
Solutions created in this section: F
solution, T solution, cell solution.
To make 1 mL of fibrin gel
(3.3 mg/mL final fibrinogen concentration, 25 U/mL final thrombin
concentration), create F solution in a conical tube, by adding 112
&l of the fibrinogen stock to 558 &l of 20 mM HEPES
Buffer in 0.9% saline solution. In a separate conical tube, create
a T solution by adding 17 &l of the thrombin stock, and 1.3
&l of 2 N Ca solution to 135 &l of DMEM. See Table 1.
In a third conical tube,
prepare a cell solution by spinning down the cells and
resuspending the cells in a volume so that the concentration of
cell is 29.4 million cells/mL or 6 times the concentration of the
desired final concentration of cells in the construct
When you are ready to cast
the fibrin gel construct, prep a 1 mL syringe with a 18G 1 ? inch
long needle. Have a 21G 1 inch needle ready as well.
The fibrin solution is
created at a 4:1:1 ratio of F solution: T solution: cell solution.
To make one mL of gel, add 667 &L of F solution into a
clean 50 mL centrifuge tube, followed by 167 &L of cell
solution, and lastly add 167 &l of T solution. Pipette to
mix solution together being careful not to introduce bubbles. Once
solutions are mixed, the reaction has started and the injection of
the constructs should be done immediately.
Take the previously prepared
syringe with the 18G needle and draw up fibrin solution. Take care
not to invert the syringe to prevent bubbles from getting into the
needle. Replace 18G needle with a 21G needle. Tap syringe gently
to force out air bubbles.
Insert syringe into the mold
between the stopper and casing following the groove in the Teflon
O-ring and inject the solution into the mold. Tilt the mold with
the groove on top to insure complete filling. Remove the syringe
and continue to fill remaining molds. Enough gel solution can be
created to fill several molds at the same time. However, because
the solution gels quickly, it is generally a good idea to limit
the number of constructs injected at a given time to 6.
Wrap the molds in Parafilm in
groups of three and place in the incubator or oven at 37° C. Allow
the gels to incubate in the molds for 20 minutes to allow the gel
time to polymerize.
Fill each culture jar
(Nalgene straight-side jar) with 21 mL of myocardial construct
medium per construct. Use the sterile 3 cc syringe casing as a
plunger to force the mandrel with the construct into a large petri
dish with DMEM. Then place the construct into the sample jar. Each
16 oz jar can hold up to 6 constructs, while each 4 oz jar can
Screw the caps on the jars
and transfer them to the incubator. Inside the incubator, loosen
the caps on the jars to allow for gas exchange.
10)After 24 hours, take a sterile
dental pick and push the construct away from the white teflon
O-rings on the ends of the ring mold to ensure uniform alignment
of the construct (See Figure 2 in representative results).
8. Analysis techniques (after 2 weeks in
culture) - contraction force testing
Solutions used in this section:
DMEM, myocardial construct media.
Clamp the alligator clips on
the lead coming from the stimulator to the wires on the electrodes
in the bath. Power up the data acquisition board, pulse generator,
and force transducer. The force transducer should be switched to
the 5g setting and zeroed. Open a custom LabView program that
displays and saves the data from the force transducer. Create a
new, empty text file in the data folder for each sample.
Place DMEM into the 37° C
water bath and allow time for it to warm up before testing the
constructs. Once warmed, place 37°C DMEM into the medium bath of
the force measurement system (see Figure 3A).
Remove the construct from the
sample jar by gently sliding the construct ring from Teflon
support with tweezers and place the construct over the fixed metal
post in the force measurement system medium bath. Do not grip the
construct with the tweezers! Rather, use the tweezers to push and
lift the construct off of the mandrel support.
Place the other end of the
construct over the transducer arm and tighten until the transducer
reads 0.50V, (approximately 1.0 grams force or 10 millinewtons of tension)
Select the text file for the
contraction force data to be recorded into.
On the cardiac stimulator
(Model # S88X, Grass Technologies), set the pulse voltage to 20V
(8 V/cm), 6ms duration, and a rate of 1 Hz.
Start the electrical pacing
by pressing the "output on/off" button
Start recording until
waveform becomes regular.
Carefully remove the
construct from the force measurement system medium bath and place
it back into culture medium. Then remove the DMEM from the force
measurement system medium bath and replace with fresh, warm DMEM
to analyze additional samples.
10)Cut the construct and unroll it
so that you can measure the length and width of the construct
11)Cut the construct into sections
to be used for additional analysis measurements including
viability, histology, or
blot measurements.
9. Analysis techniques (after 2 weeks in
culture) - Live-Dead Assay for viability (with Invitrogen
Live/Dead Assay)14 :
Solutions used in this section:
EthD-1 stock solution, calcein AM stock solution PBS
Rinse samples with 3x 5
minute washes in PBS.
Add 20 &l of 2 mM
EthD-1 stock solution to 10 mL of sterile PBS and vortex to ensure
thorough mixing.
Add 5 &l of 4 mM
calcein AM stock stolution to EthD-1 solution. Again, vortex to
ensure thorough mixing.
Add enough volume of the
above solution to cover the construct.
Incubate covered (to prevent
photobleaching of the dyes) for 30 minutes at room temperature.
Remove the dyes and replace
with warm PBS. Observe and record images with a fluorescent
microscope. Calcein is excited by 494 nm light and emits 517 nm
light, while ethidium homodimer-1 is by 528 nm light and emits 617
nm light. See Figure 4 for sample results.
10. Analysis techniques (after 2 weeks
in culture) - immunohistochemistry for important myocyte proteins:
Solutions used in this section: PBS,
4% paraformadehyde in PBS, 5% donkey serum in PBS, antibodies in
PBS, 0.1 ng/mL Hoechst 33258 in PBS.
Rinse samples with 3x 5
minute washes in PBS.
Fix with 4% paraformaldehyde
in PBS solution, for 2-3 hours at 4°C
Rinse samples with 3x 5
minute washes in PBS.
The sample can now be
embedded, sectioned and stained according to the protocol of
choice. The remaining portion of this protocol covers whole
construct staining for imaging with confocal microscopy. Note that
the incubation times are longer than that for sectioned tissue as
there needs to be more time for the antibodies to diffuse into the
construct. In addition, all of the remaining steps are conducted
at room temperature.
Add 0.1% Triton-X in PBS to
the samples for 30 minutes to permeabilize the cell membranes.
Rinse the samples with 3x 10
minute washes in PBS.
Add 5% donkey serum in PBS to
the samples for 1 ? hours to block any non-specific binding of the
secondary antibody to the samples.
Add Connexin 43 (1:50
dilution) and Myosin Heavy Chain (1:100 dilution) primary
antibodies in PBS to the samples for 3 hours. In order to label
both proteins consecutively you must make sure that the primary
antibodies are from different hosts (i.e. rabbit and mouse).
Rinse the samples with 3x 10
minute washes in PBS.
10)Add the appropriate
fluorescently labeled secondary antibodies in PBS for 3 hours.
11)Rinse the samples with 3x 10
minute washes in PBS
12)Just before imaging the samples
on the confocal microscope, add 0.1 ng/ml Hoechst 33258 in PBS for
15 minutes.
13)Rinse the samples with 3x 10
minute washes in PBS.
14)Analyze the samples expression
of MHC and Cx43 by imaging the cells with a confocal microscope
(See Figure 4 for example result).
11. Representative results / Outcomes:
The cardiomyocyte fibrin construct
initially covers the entire width of the mold (Figure 2B). No
bubbles should exist in the construct and it should look uniform
across the entire length. After two weeks of culturing, the
construct contract to approximately 1/4 of the initial width
(Figure 2C).
When the construct is electrically paced
in our custom contraction force device (Figure 3A), twitch force
data can be generated as shown in Figure 3B. The waveform can be
analyzed separately in MATLAB (MathWorks) to determine the force,
rate of contraction, and rate of relaxation. Twitch forces of
approximately 1.3 mN are expected 6 .
Cell viability of the construct is
dependent on the depth of the construct, due to the diffusion
limitations of oxygen into the construct. On the surface of the
construct, Figure 4A, high cell viability is observed. With
confocal microscopy, Figure 4B, the aligned structure of the
construct is observed due to the Myosin Heavy Chain, MHC, is
important for contraction, shown in red. Connexin 43, shown in
green, is necessary for cellular coupling between myocytes.
References:
Stegemann, J.P., Hong, H., &
Nerem, R.M. Mechanical, biochemical, and extracellular matrix
effects on vascular smooth muscle cell phenotype. Journal of
applied physiology. 98, 05).
Guarnieri, D., et al.
Covalently immobilized RGD gradient on PEG hydrogel scaffold
influences cell migration parameters. Acta biomaterialia
Krebs, M.D., et al.
Injectable poly(lactic-co-glycolic) acid scaffolds with in situ
pore formation for tissue engineering. Acta biomaterialia
Zimmermann, W.-H. et
al. Engineered heart tissue grafts improve systolic and
diastolic function in infarcted rat hearts. Nature
medicine . 12, 452-8 (2006).
Chung, C. & Burdick, J.A.
Influence of Three-Dimensional Hyaluronic Acid Stem Cell
Chondrogenesis. Tissue engineering. 15 (Part A), (2009).
Black, L.D. et al.
Cell-induced alignment augments twitch force in fibrin gel-based
engineered myocardium via gap junction modification. Tissue
engineering. 15 (Part A), 09).
Syedain, Z.H., Weinberg, J.S.,
& Tranquillo, R.T. Cyclic distension of fibrin-based tissue
constructs: evidence of adaptation during growth of engineered
connective tissue. Proceedings of the National Academy of
Sciences of the United States of America. 105, 08).
Falvo, M.R., Gorkun, O.V., &
Lord, S.T. The molecular origins of the mechanical properties of
fibrin. Biophysical chemistry . 152, 15-20 (2010).
Jockenhoevel, S. et al.
Fibrin gel - advantages of a new scaffold in cardiovascular tissue
engineering. European journal of cardio-thoracic surgery :
official journal of the European Association for Cardio-thoracic
Surgery . 19, 424-30 (2001).
10.Ryan, E. Structural Origins of
Fibrin Clot Rheology. Biophysical Journal . 77,
11.Williams, C. et al. Cell
sourcing and culture conditions for fibrin-based valve constructs.
Tissue engineering . 12, 06).
12.Grassl, E.D., Oegema, T.R. &
Tranquillo, R.T. A fibrin-based arterial media equivalent.
Journal of biomedical materials research. 66 (Part A),
550-61 (2003).
13.Barocas, V.H. & Tranquillo,
R.T. An anisotropic biphasic theory of tissue-equivalent
mechanics: the interplay among cell traction, fibrillar network
deformation, fibril alignment, and cell contact guidance.
Journal of biomechanical engineering . 119, 137-45(1997).
14.LIVE/DEAD Viability/Cytotoxicity
Kit *for mammalian cells*. Small (2005).
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