Fixatives - Science topic

Rana Hamza Shakil a message has been sent

Do you have the proper potential for the structure that you are using?

Also, just FYI min_style should come before minimize command, it doesn't matter over here as the default is "cg".

You can try removing the box relaxation at the start.

I think improper potentials is the culprit for the "absurd values" you are getting.

Heating with excess tris can reverse aldehyde modifications of amino groups (imine formation), so I'd just try and set up a series of experiments e.g. perfoming PCRs or whatever you want to do with the DNA later.

Altenatives to PFA could be methanol or ethanol.

I encountered a similar error in my structural problem with TIE CONSTRAINT. In my case, this error occurred as I permitted the movement of slave nodes to adjust the position initially. Solved this error by unticking the option "Adjust secondary surface initial position" in the interaction section.

Dear Naima Ghemmit-Doulache ,

thank you so much for your answer! I understand my SWV peak now... Do you know if there is some way to fix this issue?

If you mean to correct the titles of your papers in ResearchGate, see "How do I edit my research item's details?" in https://explore.researchgate.net/display/support/Reviewing%2C+featuring%2C+and+editing+your+research for instructions.

Please note that you wrote to the RG community, not to the RG team / administrator. However, they will not help directly, because you can make the corrections yourself.

Hi,

It is unlikely that you will come across research papers describing the fixation of cerebral organoids using paraffin. I attempted this approach in the past, but encountered difficulties. Firstly, cerebral organoids are already small tissue structures, and dehydration with alcohol further reduces their size. Secondly, when I attempted to embed the organoids in paraffin, they simply vanished. I could not observe the organoids in the resulting sections. Thirdly, even if successful in preserving the tissue, I would undoubtedly lose a significant number of slices.

Therefore, I recommend employing a different method for your work, specifically using a mixture of OCT (optimal cutting temperature compound) and 30% sucrose (in a 1:1 ratio) to freeze the organoids and subsequently slice them using a cryostat.

I hope this information proves useful to you.

For anyone reading in 2023, there is a solution coming soon from the startup I work at (LASE Innovation). We are commercializing instrumentation and Laser Particles for single-cell barcoding that enable you to acquire repeated flow measurements of the same cells. This lets you measure your samples' fluorescent signals at multiple points throughout a harsh processing protocol (before and after a harsh perm) at the points in time where signals are at their maximum intensities. The Laser Particles act as unique barcodes for the cells, and so your data is stitched together between measurements for every single cell. You end up with one FCS file that appears as though you measured everything at the same time. We recently showed at ISAC's CYTO conference in Montreal this recovers original GFP fluorescence through the harshest fix/perms (FOXP3 kit) in a case of intracellular cell cycle staining, outperforming anti-GFP staining. We also demonstrated the full recovery of fluorescence due to damaged antigen epitopes and degraded fluorochromes through a methanol perm in a phospho-flow protocol. If you are interested in becoming an early adopter please get in touch :-)

I had a similar issue. Are you getting PDF files as well? Because Adobe is one of the main programs on my computer and I could find out that the .pdb files were considered to be a PDF and opened with it. But when you choose to open with Chimera it works.

Find impedance for given voltage by measuring current, Z= V/I, then find resistance using DC source, R= Vdc/Idc. After finding Z and R could find reactance X = Sqarroot of ((Z)(Z)-(R)(R)), inductance L =X/2(pai)(f), where f the frequency…..

Hi Alexandre,

Yes you can, we used it in our lab and got a good signal of PI in a cell line. Make sure to add antifade.

@all Paul Salib Hello! I apologize for any confusion caused. When I mention PCR test, I am indeed referring to the RT-PCR (Reverse Transcription Polymerase Chain Reaction) method, which is commonly used for amplifying RNA sequences. It is typically employed to detect and quantify specific RNA targets, such as viral RNA in the case of diagnosing infectious diseases.

Regarding your question about assessing the suitability of amplicons for sequencing, running a gel-electrophoresis can be a helpful step. Gel-electrophoresis allows you to visualize the amplified PCR products and determine their size range. By comparing the band sizes with a DNA ladder or marker of known sizes, you can estimate the length of your amplicons.

This information can be useful before proceeding with sequencing, as it helps to ensure that the amplified fragments are within the desired size range for your specific sequencing method. If the amplicons fall within the expected size range and have sufficient quantity, it increases the likelihood of obtaining successful sequencing results.

Therefore, running a gel-electrophoresis prior to sequencing can be a good practice to assess the suitability of your amplicons and ensure they meet the requirements for your sequencing experiment.

Gary James Hunter Thanks for you reply.

Thanks for your valuable comments

Be careful when using meta-GGA. The default grid of ORCA is not fine enough to compute the Laplacian of the electron density.

Which version of abaqus you use?

1) At first , you have to create the model by defining the properties and geometry of the model.

2)Then mesh the model and apply the appropriate boundary conditions to simulate the loading and constraints in your analysis.

3)After this for adding a deflection expression, you can use the Abaqus Expression Language (AEL) or Python scripting.

4)You can create an expression by navigating to the "Model" module and selecting "Expressions" from the drop-down menu.

5)Then in "Model" module, select "Output Requests" and create a new request such as displacements or strains, and set the "Component Scope" to the desired direction of deflection. In the "Expressions" section of the request, assign your deflection expression to the appropriate variable.

6) Then just run the analysis and post processing for extract the deflection result.

I think these steps helps you to solve your problem.

Kika VI

I have revised the lead text (Mathematics and Causality: A Systemic Reconciliation). Now it has become 8 pages in A-4. See the link below.

I believe your questions may be answered here in my humble manner. Further questions and suggestions are most welcome. Thanks.

You're welcome, Maria Fernanda Rosado! I'm glad to hear that my answer was useful to you. If you have any more questions or need further assistance, feel free to ask.

Shaina Selles Hello! did you find any way to solve it?

It was working fine for me until I added sequences and it gave me the length error, but I don't understand it.

Thank you

thank you Joana B. Caldeira and Nikolay Klyashtornyy for your rsponses. I'll try the elution buffer Nikolay Klyashtornyy mentioned.

To extract a specific part of the flow field in OpenFOAM and start a new project on it, you can follow these general steps:

1. Identify the region of interest: Determine the specific part of the flow field that you want to extract for your new project. This could be a particular geometry, a region within the domain, or a specific time frame of the simulation.

2. Define the extraction criteria: Specify the criteria for extracting the desired part of the flow field. This can include conditions based on geometric boundaries, cell labels, time steps, or any other relevant parameters.

3. Use the appropriate OpenFOAM tools: OpenFOAM provides several utilities and functions that can help with extracting and manipulating the flow field. Some commonly used tools include:

- `patchAverage`: This utility can be used to extract averaged values on a specified patch or surface.

- `foamToVTK`: This utility converts OpenFOAM data to VTK format, which is widely supported by visualization tools.

- `sample`: The `sample` utility allows you to sample the flow field at specific locations and save the data in a new file.

- `fieldMinMax`: This utility can be used to find the minimum and maximum values of a field within a specific region.

4. Write custom code or scripts (if needed): Depending on your specific requirements, you may need to write custom code or scripts to perform more complex extraction tasks. OpenFOAM provides a comprehensive C++ library for customization and development.

5. Start a new project: Once you have extracted the desired part of the flow field, you can start a new project based on it. This may involve setting up a new case directory, adjusting boundary conditions, mesh refinement, or any other necessary modifications for your new simulation.

6. Perform analysis or simulation: With the extracted flow field and the new project setup, you can now perform your desired analysis, post-processing, or simulation on the specific region of interest.

Remember to carefully document your steps and maintain clear organization to ensure reproducibility and traceability of your work.

Same problem

Hi Arup,

Thank you for your detailed response and valuable information. I have tried methanol fixation, it seems methanol alone didn't work well, I will try methanol/acetone mix again, and will also try digitonin and blocking.

•Formalin

• Zinker Solution

• Bouin Solution

•Carnoy

thank you Mostak Ahamed for your information

That is a very specific operation. I do not think that ChIP will help you with that.

Improvement that could potentially work is a reduction in formaldehyde concentration used for crosslinking - I would try 0.01, 0.001, and 0.0001% and no longer than 5 min.

I've attached an image of how formaldehyde crosslinks proteins to DNA in 15 min of treatment. As can be seen for the 0.1% treatment almost all proteins were isolated with the DNA.

I would also try isolating nuclei first and then fixing them (I know you said that this would not be preferred) but it will give you a clear cut-off: cytoplasm/nucleus.

the different salts help precipitate the RNA but excess salts or other salts could inhibit downstream reactions.

this chapter explains it well

Hi,

I routinely do RNAscope, HCR, and classic whole mount in situ on mouse embryos, they both work very well with 24h+ fixation in 4%PFA obtained by diluting 32% PFA in 1X RNA grade PBS (made with DEPC water) without adjusting the pH.

Hope that helps,

Best,

Camille

Thank you Samuel for your time! I have studied algebraic topology and cohomology theory, and can understand most part of your answer. Thanks again for such details!

Anton E. Kulagin Thank you for your reply. I calculated an example where u''''(x)=e ^ (x ^ 2), and the boundary condition is u (± 1)=u '' (± 1)=0. I set u (x)=(1-x ^ 2) ^ 2q (x), and required q (± 1)=0. The results are different from the ones I proposed using Weidman and Reddy to validate a MATALAB differential matrix suite. So I need to think again. Thank you for your reply.

Fatih Nejdet Memiş The problem is caused by the language settings on the computer. Probably your system language is Turkish. Changing the system language to English will fix your problem.

Kind regards

It depends on the purpose of your experiments. There are several ways, but they all have common elements. You have to fix one of the friction surfaces to a moving element (usually a disc or a swinging table) and the other to a fixed force sensor. Record the force signal parallel to the fixed surface using a datalogger. Taking into account the normal load on the fixed surface and the force signal, the coefficient of friction can be calculated. Examples of typical configurations are pin-on-disc, ball-on-disc and reciprocating.

Thank you so much. I fixed it. But I stil can't do RMSD analysis. As I understand it, the problem is in the complex_wb.psf file. But I don't know how to fix it. After opening the VMD program, I load the complex_wb.psf file and then I load the .dcd formatted file. It gives an error like Unable to molecul and in the terminal like this:

I totally agree with Urs.

Some other things that you should consider.

After the transcardial perfusion with 4%PFA (5-10min or until the liver is yellowish) you should immerse fixed them for 24h. After that wash them in tab water for 24h. Cryoprotect them (10-20-30% sucrose with azide) and cut them on the cryomicrotome and let them dry for 24h at 37deg C.

Do antigen retrieval and stain..

Good luck!

Martin

In general, the program failed to localize the TS. I don't use qst3 or qst2, it is much more input preparation to do and it does not have any advantage over opt=ts when you have a good approximation to the TS.

I suggest, what I always do, first to scan the PES:

#P DFT/basis Opt=ModRedundant

0 1

xyz

B 7 21 S 12 0.10

In this example, the bond "B" betwen atoms "7" and "21" is going to be scanned "S" through "12" steps of "0.10" angstroms step size.

Only if you obtain a smooth curve of the scan you'll know you are in the correct pathway, in that case select the structure at the maximum point of the curve and do a TS calculation directly:

#P DFT/basis Opt=(ts,noeigentest,calcfc) freq

If you don't see a smooth curve, then finding the TS becomes readly challenging, or you should look for another way to do the scan PES (other bonds, angles or even dihedrals).

Best, Pablo

Hello M.Tamil Mani Subi

If the sample is clumpy, one will not be able to readily distinguish cells of interest from the clumps they are attached to. Therefore, sample preparation becomes the critical first step in any flow cytometry experiment.

I have mentioned a few suggestions below which you could follow on how to avoid cell clumping.

1. The dead cells release DNA in the medium. DNA being sticky in nature can form cell aggregates by causing cells and debris to stick to one another forming clumps. If cells appear clumpy, calculate the volume of DNase I solution that should be added to the sample to yield a final concentration of 100 μg/mL DNase I. Add DNase I solution dropwise to the cell suspension while gently swirling the tube. Incubate at room temperature for 15 minutes. Wash the cells with culture medium or buffer containing 2% FBS. Gently invert to mix, then centrifuge at 300 x g for 10 minutes at room temperature. Discard as much of the supernatant as possible, then gently resuspend the pellet. If cells still appear clumpy, then you will have to pass the sample through a 37 - 70 µm cell strainer into a fresh conical tube. Rinse the sample tube three times with the culture medium or buffer containing 2% FBS, then pass through the strainer.

2. You need to know that calcium and magnesum can promote cell clumping. Use Ca++ and Mg++ free PBS for your staining buffer, and consider adding 1mM EDTA to your buffers as well.

3. If you pellet cells too hard, they can clump. Ensure that cells are not subjected to high centrifugal force.

Hope these above suggestions may help you get rid of cell clumps.

Best.

The error you are facing seems to be related to a syntax error in the file "/opt/python/3.7.12/lib/python3.7/site-packages/scipy/init.py". It is possible that this file is corrupted or has been modified. You can try to reinstall scipy package to fix this error. You can do this by running the following command:

Copy codepip uninstall scipy pip install scipy

If this does not work, you can try installing an older version of scipy package by specifying the version number:

Copy codepip install scipy==1.7.1

Also, make sure that you have installed all the necessary dependencies and libraries required for running bandup. You can refer to the documentation of bandup for the list of dependencies required.

Assalamo Alaykom Dr. Mohamed,

Asphalt cracks happen due to different reasons depending on the cause of the crack, for example, Transverse Cracks that you can see across the road (transverse direction) and also the Block Cracks that you can see the road is divided into big blocks could be possibly according to Thermal Stresses. and this type of stress is produced in the asphalt pavement (expansion and contraction of the binding bituminous material) due to the big difference in temperature between night and day, and also the different seasonal temperature, you can see it clearly in Upper Egypt.

Another reason for cracks is Fatigue due to overloading causing a famous type of cracks called Alligator Cracks that looks like a crocodile back. Another type of cracks are the Longitudinal Cracks, These types of cracks predominately created between the existing pavement and new widening but may also be a result of expansive clay subgrade soils.

Regarding the Preventive actions, there are many actions depending on the density and severity of the crack, starting from Sealing and Filling of the crack with a a material that will fill the crack preventing the water (The most dangerous Asphalt pavement Enemy) from getting inside the pavement layers causing an accelerated deterioration of the asphalt pavement.

Depending on the environment you might also consider lupins

No, the size cannot be smaller than a certain amount. The error is also displayed with sentences:

(&H8000ffff) Unable to evaluate expression: "r_in" (.Create)

or

some objects dimentions are too small for 3D modeler.please set smaller units first.

It is challenging to find a cell fixation protocol that will preserve both RNA and cell surface markers, as fixation can potentially affect the integrity of both. However, there are several options that can be optimized to achieve the best preservation of both.

One potential approach is to use a fixation method that is mild and fast, such as paraformaldehyde (PFA) fixation for a short duration. This can help to preserve the RNA and cell surface markers, but optimization of the protocol is essential.

Here are some general guidelines for optimizing a PFA fixation protocol:

It is essential to note that optimization is critical for preserving RNA and cell surface markers, and different cell types and targets may require different fixation protocols. Therefore, it is recommended to perform optimization experiments for your specific cell type and surface markers.

This happens when the ratio of deformation to wave speed exceeds 1. You can modify the mesh sizes. Toggle hourglass and distortion control. See this thread:

DESS is the most commonlhy used preservative for molecular studies.

Dear Haowei,

1. Presence of several local maxima in the stress-exposure curve in a possibility that you should consider in you material model. You can find in our paper below an efficient method that was proposed to identify the global maxima and find the fracture angle with a high accuracy:

2. The fracture angle at the onset of failure should be stored as the fixed value of the fracture angle in each element. There is no need for further calculation after the damage initiation.

I had to stain rat tissues stored in PFA from 10 years ago. They turned out fine. Im not sure about the max time in PBS.

Try defibrinating or adding citrate.

I replied in your other parallel thread.

Hello Nguyen Trung Hieu,

Yes, you can fix the direction of magnetic moments of atoms in a CASTEP geometry optimization calculation. To do this, you need to set up the calculation input file (.cell file) with the appropriate settings.

Here are the steps you should follow:

Define the initial magnetic moments for each atom:

In the .cell file, specify the initial magnetic moment of each atom using the "spin" keyword in the "%BLOCK POSITIONS_FRAC" or "%BLOCK POSITIONS_ABS" section. For example:

===================================

%BLOCK POSITIONS_FRAC

Fe 0.0 0.0 0.0 spin=2.0

Fe 0.5 0.5 0.0 spin=-2.0

%ENDBLOCK POSITIONS_FRAC

===================================

Then set the "fixed spin moment" option:

To fix the direction of magnetic moments, you need to use the "fixed_spin_moment" keyword in the "spin" section of the .param file. This keyword ensures that the direction of the magnetic moment is fixed during the geometry optimization.

Here's an example of how your .param file should look like:

===================================

task : geometryoptimization

xc_functional : PBE

cut_off_energy : 400 eV

spin :

{

polarized

fixed_spin_moment

}

===================================

This will ensure that the direction of the magnetic moments of the atoms is fixed during the geometry optimization process. Note that the direction is fixed, but the magnitudes of the moments can still change during the calculation.

Regards,

Alessandro

I am facing same issue

If you have resolve it please tell me

Sure Sir ....

I will look into them and will let you know.

Thank you

Dear Louise Muller ,

It might be something temporary. I check here https://www.researchgate.net/profile/Louise-Muller-2/publications and clicked on conference papers (which indicated (1)) and numerous conferences papers appeared. I would say just wait and see for awhile and otherwise follow the advice given by Wolfgang R. Dick

Best regards.

Why not do the experiment? Measure a fresh tissue, fix it (PFA), measure again.

But to answer your question: The closer you are to the living state the better, scanning in buffer would be the best. The ECM is fairly robust in comparison to other structures, I would assume that unfixed will give you the most accurate stiffness measurements. Once you aldehyde-fix biology a lot of unpredictable things happen (tissues turn brittle, proteins are crosslinked but nothing else), and solvents are even worse because a lot of material gets extracted. Also keep in mind that NBF contains methanol. And all these parameters vary depending on which tissue you're looking at. You'll have to do the experiment! ;)

For general structural preservation PFA>NBF>solvents

! B3LYP cc-pVQZ opt

! KDIIS DAMP SOSCF LSHIFT rijcosx

* xyz 0 1

C -0.570184 -0.027415 0.061066

H -1.059981 -0.677797 0.786241

H -1.071556 -0.118316 -0.902852

C 0.899274 -0.433274 -0.083437

H 0.956453 -1.466772 -0.426156

H 1.406977 -0.350205 0.878788

H 1.395391 0.209840 -0.812005

O -0.657301 1.137419 0.447493

H -1.299076 1.726520 0.050861

*

But I must say right away that the B3LYP functional incorrectly estimates the LUMO energies.

Yes, and the basis set for LUMO is needed with the addition of diffusion functions.

Thanks Mite, Much appreciated!

Best, Ivan

Remove the guard column and re-test. Does the pressure drop back down to normal ? Keep it simple. If the guard column causes the pressure to increase by more than 5%, it has either been installed incorrectly, is the wrong type or something is partially obstructed. Do not change the flow rate to achieve a specific pressure value. HPLC does not work that way. Pressure is a measured change, not a method development variable.

If you are running isocratic, then the pressure should be stable. If you run a gradient with MeOH, then the pressure would be expected to change. For more info: Please contact someone at your school how has practical training in using your HPLC system. Someone local to you can help you troubleshoot these types of basic problems on-site with ease. Ask for help. A web forum is a poor resource in this application.

This STK import process requires some manual effort. Moreover, OPNET 14.5 support is very limited now. However, you may try the following steps.

Subodh Choukidar You might resolve your issue. Actually, I am facing a somewhat similar error. I am working on a Ca-complex. I have used Gaussian-optimized geometry using a 6-31G(d)/B3LYP basis set. Avogadro software was used to open a .log file for converting it into a .mol2 extension. The optimization was normally terminated by Gaussian. Still, I found an error while uploading the file both into the CGenFF server and CHARMM-GUI.

The error states that:

readmol2 warning: non-unique atoms were renamed.

Now processing molecule CMEL ...

attype warning: element not supported;

skipped molecule.

I am a newbie in molecular dynamic simulation. Kindly suggest if CGenFF supports Ca(2+) or Zn (2+) or how can I modify my file to make it compatible.

Hello. This problem has also troubled me for a long time. In lsdyna, the none reflection 2D boundary conditions are required to be given in counterclockwise order. You need to check whether the boundary nodes are arranged in the set node you defined in counterclockwise order. You can take a look at the last picture of this boundary condition description in the manual document, I believe it will inspire you. The image below is a screenshot from the manual.

To resolve this error, you can follow these steps:

By ensuring that the keyword cards for step-dependent input appear after the first *step card, you should be able to resolve this error and run your Abaqus simulation successfully.

Of course, the value of housing fluctuates. So does the value of stocks. But they may not fluctuate in synchronization with each other. My impression is that real estate is usually the first to drop in a recession. In the US right now, real estate is in decline (there are, of course, regional exceptions).

To get a blue Prussian Blue pigment there has to be Fe3+ in your cells. Iron tends to be washed out in tissue during fixation in aequous solutions. The HCl in the Prussian Blue-mixture liberates Fe3+ out of compounds like Ferritin. These have to be captured by the Hexacyanoferrat to produce the blue pigment. In tissue-staining the insoluble pigment is captured within the tissue-architecture. I don't know, if minute amounts of Iron in a cell can be detected in this way.

I assume, that the brown staining is just an uptake of Hexacyanoferrat to your cells.

Any new suggestion?

Dear Alpesh Makwana

I am facing the same issue.

I am using UEL, it works on a single processor but not on multiple CPUs. I tried different versions of Abaqus on other systems, but the result is the same.

I was wondering if you could help me out!!

Thanks,

Mohammad

There are several reasons why a peak may appear broad in HPLC, and changing the concentration of the mobile phase can certainly have an impact on peak shape. Here are some possible ways to fix broad peak shapes in HPLC:

This error message typically appears when there is a conflict between the citation styles used in your document and the Mendeley software. Here are some steps you can take to resolve the issue:

✓ Check the citation style: Make sure that the citation style used in your document matches the style in Mendeley. If there is a discrepancy between the styles, it may cause the error message to appear.

✓ Remove the index area: If the error message persists, try removing the index area from your document. To do this, go to the "References" tab in Word and click on "Insert Index". In the dialog box that appears, select the "Index" tab and click on "Delete Index". This should remove any existing index areas in your document.

✓ Re-insert citations: Once the index area has been removed, try inserting your citations from Mendeley again. Make sure that the citations are placed in the correct location in your document and that they are formatted correctly.

Regards,

Do you know the chemical formula of the crystals? if yes you can chek in some places what are iits solvents.

This is easier to address if we know what kind of engine you are working on. I am going to assume you are working with a 4-stroke engine but is it an inline 4 cylinder, inline 6 cylinder, V-6 or possibly a V-8.

Inline 4-cylinder engines very often use a 'flat plane' crank with cylinders 1 and 4 traveling in unison and 2 and 3 in unison. This lends itself well to a 1-3-4-2 firing order. (notice an outside-inside-outside-inside pattern) There are a few oddball cranks but this covers 98.5% of them. A drawback to the 4-cylinder flat crank it that two pistons are at TDC when two are at BDC so all four pistons are changing direction at the same time.

Inline 6-cylinders have 1-6, 2-4, 3-4 travelling in pairs with the pairs separated by 120 degrees on the crank. Firing order is universally 1-5-3-6-2-4. I expect there is an inline 6 out there that does not follow that rule but in 45 years I have not seen it.

V6 it gets a little more confusing at the engines can be either 60 degree or 90 degree Vees each having advantages and disadvantages. Different manufacturers also number their cylinders differently making a standard arrangement more difficult to simply state.

V8 are a little more standardized than V6. Most V8 use a 90 degree crankshaft on a 90 degree V. General motors tends to number their cylinders 1-3-5-7 front to back on the left side and 2-4-6-8 front to back on the right side. There are alternative firing orders but a very common on with that arrangement is 1-8-4-3-6-5-7-2. You have to draw it out to see how the cylinders 'hit' side to side and front to back. There are some other firing orders out there but that is most common. With the theory that various first and second order harmonics are better controlled, regardless of how many cylinders you have.

In all cases, the cam has to be ground so that the valves are behaving appropriately for whatever firing order you are working with.

So, yes, way down in engine design there are some guidelines for setting up a firing order. I have never seen a specific formula for creating a firing order mostly guidelines as very few of us are going to be able to just go whip up a different crankshaft because we feel like trying something.

Simulated Body Fluid (SBF) is a solution that mimics the ionic composition of human blood plasma, and it is commonly used for bioactivity tests to evaluate the performance of biomaterials, such as implants or scaffolds.

To synthesize SBF, the following steps can be taken:

If your synthesized solution has a high pH of 8 or above, it suggests that the pH adjustment step was not done correctly. To fix this problem, you can add a small amount of HCl to lower the pH to the desired range of 7.2-7.4. You can then use a pH meter to verify the pH and make additional adjustments as needed. It is important to note that the pH of SBF is critical for its bioactivity and should be carefully monitored and adjusted.