Oocytes - Science topic

For your hyaluronidase step, do you use a supplemented PBS or just plain PBS? In human IVF we use mHTF (with HEPES) for that step on a warm (37degree surface) Osmolarity very important.

Hello Yogesh,

Membrane blabbing is one of the morphological features characteristic of apoptosis commonly observed under in vitro culture conditions in various somatic cell types as well as in germ cells like oocytes under stressed or drug-induced conditions.

Hypertonic treatment to the Xenopus oocytes could induce a osmotic stress as the osmolarity of Xenopus oocytes is normally 240mOsmol. The hypertonic conditions will generate the hypertonic shock that may induce in the reduction of size due to oocyte shrinkage involving exocytosis and the other related to an ionic current. For detail see the link given below:

Release of ATP induced by hypertonic solutions in Xenopus oocytes. J Physiol. 2003 Feb 15; 547.

Membrane blabbing are seen in oocyte undergoing shrinkage during apoptosis in experimentally induced conditions and apoptosis under in vitro culture conditions.

Although we have not generated membrane blabbing in xenopus oocytes but we have observed membrane blabbing in rat oocytes undergoing apoptosis under in vitro cultured conditions.

The sequential development of membrane blabbing in rat oocytes cultured in vitro conditions can bee seen along with the detail protocol in our published paper link given below:

Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 2005; Aug;10(4):863-74.doi: 10.1007/s10495-005-0367-8.

Hope this helps

Best

Hello Andrew Gipson,

In my opinion, it could be heterogeneous distribution of yolk platelets in the vegetal pole of Xenopus laevis oocytes. Two types of primordial yolk platelets (I and II) have been discriminated. After membrane fusion these precursors can be completely incorporated into the main body of existing platelets, numerous yolk crystals then merge and form one uniformly stratified core.

The yolk platelets ofXenopus laevis have been studied by thin-section and freeze-fracture electron microscopy to characterize the boundary membrane during yolk formation. Lipid droplets are tightly attached to the membrane at all developmental stages of yolk platelets. A direct connection of endoplasmic reticulum to the membranes of yolk platelets has not been observed. The proteinaceous yolk platelets tend to fracture along their periphery through the superficial layers.

For more detail see the following link:

Yolk organelles and their membranes during vitellogenesis ofXenopus oocytes. Roux's archives of developmental biology 198, 92–102 (1989).

Fluorescence lifetime imaging reveals heterogeneous functional distribution of eGFP expressed in Xenopus oocytes.

In this paper please see the figure 1B, similar to your attached image.

Best

Dear Omar,

do you got the answer for this question? I've meet the same situation and tried to find the explanation.

Thanks.

As I have already replied that the ovarian tissue containing follicular oocytes are better tissue for cryopreservation. And clarified a difference between oocyte and ovum. Normally oocyte is a diploid and collected only from ovary. However after ovulation, ovum is a haploid female gamete and posses first polar body.

Few basic and important aspects of oocyte biology:

Oocyte is a large specialized germ cell that is arrested at diplotene stage of meiotic cell cycle within the follicular micro-environment. These oocytes are not yet matured in the ovary and subjecting them to cryopreservation process is risky. Due to its large size (about 100 micrometer in dia) there are several possibilities of its deterioration and oocytes may become fragile due to cryopreservation that could results in the loss of its quality.

Although ovum cryopreservation is done and IVF is conducted. In spite of having a significant advances in ART till date, the IVF rate is very poor due to several limiting factors. One of the major factor is the ovum factor. Normally in Fertility clinics, ovum is collected from stimulated ovary of infertile female. In the ovarian stimulation protocol, oocytes are pushed out due to supra physiological doses of gonadotropins (FSH & LH) or by ovulation inducing drugs like Clomiphene citrate and other similar drugs. These supra physiological doses of gonadotropins and ovulation inducing drugs like Clomiphene citrate and other similar drugs may have several adverse impact of the ovum quality as stated above by Dr. Elizabeth Rex citing ASRM quote and our animal studies published in ASRM Journal Fertility Sterility :

Chaube et al., Estradiol protects clomiphene citrate–induced apoptosis in ovarian follicular cells and ovulated cumulus–oocyte complexes. (Fertil Steril
2005;84(Suppl 2):1163–72. ©2005 by American Society for Reproductive Medicine.

Clomiphene citrate inhibits gonadotropin-induced ovulation by reducing cyclic adenosine 3=,5=-cyclic monophosphate and prostaglandin E2 levels in rat ovary. Fertil Steril
2006;86(Suppl 3):1106 –11. ©2006 by American Society for Reproductive Medicine

Melatonin protects against clomiphene citrate-induced generation of hydrogen

peroxide and morphological apoptotic changes in rat eggs. European Journal of Pharmacology 667 (2011) 419–424.

There is a big difference in naturally matured ovum collected after normal cycle of ovulation and ovum collected from stimulated ovary in terms of its competency. Hence to retain oocyte quality as good as naturally ovulated one, its important to take a piece of ovary containing follicular oocyte and cryopreserved. These tissues can be thawed and oocytes are collected and cultured in maturation medium so that the fist polar body is extruded (one of the most important oocyte function) in IVF Labs/Centers. After observing the first polar body, these ovum could be used for various ART programs (including IVF).

Thanks Prof. Omer Ucar for recognizing the quality issues of the oocytes. The scientific community need to generate the potential biomarkers that could be useful for the development of "oocyte function test". The experts in the field (you, me and others) should come forward to address this issue in order to develop oocyte function test (using non-human mammalian species) for the determination of oocyte quality prior to their use in ARTs.

Hope you are interested in culture of mammalian oocytes/ovum in vitro. If yes, let me tell you that there are several culture medium available for oocyte in vitro for various purposes. For oocyte maturation, you can use M-199 (Sigma, HiMedia and several other companies), for fertilization (M-16, M--2 etc.) and embryo culture (several). All companies provides the ingredients of the culture medium and these media are provided both in powder form (to be reconstituted) as well as in liquide (ready to use) form with several options of buffering and alterations for in vitro culture such as Ca+2/Mg+ supplemented/free, etc. Actually you have to decide based on the source, stage and need of experiments and accordingly use the culture medium. Good luck

In vitro culture conditions never meets the body environment (in vivo conditions). Therefore, once the oocytes are retrieved for IVF purpose, they are always exposed to stress conditions (due to minor changes in physical, temperature and culture medium). Oocyte is more susceptible to the stress conditions due to larger surface area (100-120 microM in dia) and even minor stress conditions may initiate downstream signaling pathways to induce oocyte death under in vitro culture conditions. We have identified some morphological features that could be used to identify the stressed oocytes in vitro. The series of morphological changes that are identified in oocytes cutlured in vitro are:

1. The smoothness (clear) of oocyte cytoplam is reduced

2. Initiation of cytoplasmic granulation

3. Changes in Zona pelucida histoarchitecture

4. Polar body roughness (fragmentation)

5. Cytoplasmic dia shrinkage (more gap between zona and corona)

6. Membrane blabbings

7. Cytoplasmic fragmentation

and many more........

For more details, kindly see few of our published papers:

1. Apoptosis, 10: 863-875. IF=4.677

Good luck

Thank you Dr. Ke-Hui Cui M.D., Ph.D. for your personal opinion on introducing a new concepts of Hereditics, Cytohetics and Epicytohetics and considering both egg yolk and egg white as hereditary material. We know that the cytoplasmic inheritance (specially through mitochondrial genome) play important role in determining the fate of the individual (in mammals). I would not comment on your new concept and feel that it is premature to say anything on these new thoughts.

Are you trying to isolated the GV stage oocytes for in vitro cultre and analysis? or trying to isolate them and use for biochemical/histological analysis. There is different protocol for different targets. Kindly clarify your purpose.

Please read MRT syndrome in Https://www.hereditics.net. MRT syndrome is a new kind of artificial cytoplasmic hereditary diseases, shortened as Cytohetic diseases.

Thanks Prof. Yavuz Öztürkler for your reply. Our findings also supports your views. But the role of Cdk1 activation due to phosphorylation/dephosphorylation on specific amino acids sequences are seems to be important (as reported in other mammalian species).

Your question is bit confusing. From your question, it seems that you want to isolate and denude diplotene arrested immature oocytes from primordial/primary follicles of mouse ovary and to study the gene expression in denuded oocytes cultured in vitro.

If it is so, then remember that due to larger in size as compare to other cell types in ovary, immature oocytes are more susceptible for any minor changes under in vitro culture conditions. These minor changes such as temperature, culture medium, addition of hyaluronidase or trypsin will have negative impact on the oocyte in vitro. Addition of hormones like hCG etc. may also have influence on the gene expression analysis of oocytes due to presence of encircling somatic cells.

You have to be very careful, while isolating and denudation of immature oocytes in vitro in terms of addition of either enzyme or hormones (both exposure time and concentration).

I would suggest you to reduce the diameter of micro-glass pipette, and keeping all physical and biochemical factors into consideration, make repeated pipetting in order to denude the immature oocytes. Yes, it is difficult task but not impossible for a technically skilled scientist.

Hope, you would be able to do it and best of luck

Yes. It is matter of concern and although alternative methods are available in order to prevent oocyte damage during handling in vitro. The alternative methods have some or other type of negative impact on the oocyte. The development of an improved method/materials that can replace glycerol and prevent damage under experimental condition is required. To the best of my knowledge no such appropriate wax cushion is in the market which can be better in handling oocytes in vitro or for the assessment of maturation process in mammalian oocytes for confocal microscope.

Good morning, I understand your interest for the best knowledge for high technology to FIV. It should be necessary to teach at the woman (before her hormonal stimulation and intervention) the knowledge of the natural signs of fertility, as cervical mucus for maximum days of fertility and basal temperature records to diagnose corpus luteum (less than 10 days of high level is insufficient CL, 10 days are sufficient CL, 16 days are maximum natural CL, 20 days diagnose a pregnancy.) If you are interested in more information about sympto-thermal method you can consult CaMen in Milan. All teachers of STM have cases of conception after unsuccessful FIV, but not all people conceive. It should be better to learn those signs before high technology. Regards

Glutamic acid + Cs-hydroxide

Hi Franco,

I hope things have already worked on your side.

I went through the same issue from March to May. I tried to adjust all the parameters I could think of (bought new frogs, tried a couple of collagenase and concentrations, remove follicular membrane manually, different types of solution, glass vials/falcon tubes, temperature, time, autoclaved everything, etc) but nothing worked. But after we entered June, the problem just disappeared by itself by using our original protocol. I can only say that the "bad period/good period" thing is real. We sometimes just have to be patient.

Leonardo, you can verify basic things like osmolarity and pH, if the gas mixture was changed recently, any expired reagent. If you use oil on your culture, this might also be a problem (we had problems with different lots of oil). During the years I experienced all kinds of problems that resulted in the embryos dying, from CO2 problems, expired reagents, external contamination with cleaning substances... If you can provide more information about any possible changes that occurred in your system, that might help to identify the problem.

Hello Matej Murín,

The chi² test of independence and the null hypothesis is that there is no difference in the distribution of responses to the outcome across comparison groups. This is often stated as follows: The outcome variable and the grouping variable (e.g., the comparison treatments or comparison groups) are independent (hence the name of the test).

Independence here implies homogeneity in the distribution of the outcome among comparison groups. One of the limitations of chi² test is that all participants measured must be independent, meaning that an individual cannot fit in more than one category. If a participant can fit into two categories a chi-square analysis is not appropriate.

Another limitation with using chi² test is that the data must be frequency data. For example if you are just counting how many embryos show (or not) protein expression in two separate conditions, than a chi² test is appropriate. Also when calculating the number of expected individuals for each class, there should be greater than 5 for each class (and not null) for the most appropriate use of chi² test.

One last consideration is that the chi² test statistic is sensitive to sample size. Most recommend that chi² test not be used if the sample size is less than 50 (so your 30, 28, 37 and 36 oocytes in experimental groups seems too low). If you have a 2x2 table with fewer than 50 cases many recommend using Fisher’s exact test.

Hoping that these statistical informations will help you in your data analyses.

Ludovic Dumont, Ph.D.

I think you can do

Hi Melanie,

That picture or the shed part you see in your image is called as Chorion. If you follow the time span of how the eggs develop over 3-4 days you will see a very thin membrane that covers your egg. These are basically protective membranes that shed off by themselves as they grow. However, due to technical handling or shaking or any such issues the eggs may loose these membrane and you will have tiny transprent embryos in the medium (watch for them closely if dont want to loose it). Simply, you can assume it to be basically a shell protecting the main embryo. Refer Zebrafish book and you will learn more about it. If you are a beginner I would suggest to read through the questionnaires provided at the end of the book too. It will be way too helpful for you for maintaining and harvesting your egg cultures from zebrafish.

Yes! Please see the RG links below.

It is very difficult to avoid the contamination in open pulled straws, however, can be minimised by low pressure long time autoclaving i.e. 5-7 psi for 20-25 minutes.

this link could be useful:

From this possibility you can influence the amount of M2

Dear Alexandra Pineda-Muñoz

Everything is very simple. They are prepared in the same way as mammalian eggs. Deep freezing takes place in liquid nitrogen. There is also an article, unfortunately in Russian.

Regards, Sergey Viktorovich Pushkin

Its the best option. However, I'm looking for an alternative to collect both GV and MII oocytes at once because there are few animals available now. I'll give it a try and comment here later.

If it doesn't work, I'll collect GV and MII separately. Thanks Mukesh Kumar Gupta !

You need stereo microscope with diascopic light stand.

Agree with both, we reuse them for research/training purposes only, with no problem. We wash with sterile MilliQ water followed by ethanol and then UV it for 30 min before use.

After a long time, I got only one answer. But, I want membrane thickness, not a vitelline layer.

Thanks for your reply.

Yes there will be variance sir

Xiujuan Tan Hi there, I asked this question during my master project, so I am not doing this experiment anymore. If you wish to get into contact with that lab let me know.

The reason I said gdf9, because gdf9 and bmp15 comes from oocyte and control the coc complex morphology and function

Thanks Wasim Al Shebli for your feedback. You are right, ribosomes might play a role in this.

I TRIED ONCE USING FLUCONAZOLE, BUT I COUNT GET RID OF YEAST ...

We never attach oocytes and I doubt that it may be useful. For patch-clamp you will anyway use excised (most probably, outside-out) patch.

We are using chambers with a bath for oocyte where seal is formed and patch excised, and near it a flow bath for measurement. So, after excision the pipette with the patch is transferred (slowly!) into this flow bath, and oocyte is not interesting for you anymore.

In 2-electrode clamp experiments oocytes are also not attached, just help by electrodes themselves.

Please have a look at the below given RG links.

Hi!

I used Trizol and similar reagents many times to extract RNA from small samples (about 2 mg of tissue). Always cleaning at least twice with 75% ethanol before elution. After total RNA extraction, I usually purify the RNA with the RNEasy MinElute CleanUp (Qiagen) kit. I had some sample loss, but nothing to be afraid of and enough to make RNAseq. If you use this approach, clean the columns twice with the RPE buffer.

The other alternative would be to use the appropriate kit suggested by the colleagues above, or RNeasy Plus Micro Kit (Qiagen).

Best Regards!

Follow

@Schmitt

thank you for your specific answer. I agree with your comment that it is neccassary to check whether the Ab works in ICC by KO cells or overexpressed cells, when it has never been tested. However, I think it would be better to find references as I have some limitations for gene modification.

The original reference for the construct is: Liman ER, Tygat J, Hess P (1992) Subunit Stoichiometry of a Mammalian K+ Channel Determined by Construction of Multimeric cDNAs. Neuron. 9:861-871. For more detailed information on the sequence you can check maps of constructs derived from this vector, for example: http://www.euroscarf.de/plasmid_details.php?accno=P30652.

I can advise you to read "Correlation between germinal vesicle and oocyte development in the adult Japanese quail ( Coturnix coturnix japonica)

J. Embryol. exp. Morph. vol 29, 1, 145-157, 1973.

With kind regards, Marc Callebaut.

Many thanks!

Hello Riddhi Kirit Pandya,

First of all, what do you call "controls" for RT-qPCR? Housekeeping genes ?

Indeed, results (the relative expression level of each gene) need to be normalized by two housekeeping genes (and not usually only on one); as recommended by the MIQE guidelines (Bustin et al., 2009) with the use of geometric mean to generate an accurate normalization; available at http://www.rdml.org/miqe.php.

Then, be careful to evaluate RT-qPCR results visely (fold-change, relative expression, the 2^(-ΔΔCt), etc...). After computing your RT-qPCR slopes & curves data you obtain your 2^(-ΔΔCt) data. During this procedure, you have to normalise all your data on housekeeping genes (a couple) and on a "control experiment" (associated as the "1:1" value) to compare them as ratio-values: data are presented as relative expression of your genes. --Be careful to use geometric means at this step!--

If you want more more precise and detailed informations, please see Livak and Schmittgen (2001).

Try 0.5mg/ml pronase in PBS and leave your cappilaries for 20 mins, then rinse with distilled water for flushing a couple of times before using.

Works in bovine oocyte manipulation. Hope it works

We published all techniques of mouse reproductive technology on our website (CARD, Kumamoto University). Please check the superovulation protocol via following link:

The first figure in your question is from my article

As stated, the slides were stained with CREST serum (red), which stains centromeres, and anti-SYCP3 (green), which stains the axial/lateral elements of the synaptonemal complex. Staining the centromeres will help you identify the differences between the 5 substages of Prophase I.

In Leptonema, there are many centromeres and the chromosomes are very thin. In lack of better terms, it's a mess. In a zygotene cell, the chromosomes are more distinct. By the pachytene stage, there should be 20 chromosomes (for murines) and the centromeres are at the ends of each chromsome. In diplonema, you see the ends of the chromosomes appear like zippers or split ends. In diakinesis, the chromosomes are no longer present, hence little specks.

Hope this helps.

Thanks so much! I’ll give it a try. How much do you administer your mice?

thank you very much for your advice

Jiří Šichtař thank you for your helpful insight!

You have cumulus clump and oocyte has been removed. I will suggest rupture the cumulus in smaller clumps with fine needles (27Gz attached with a syringe), Place smaller clumps on hemacytometer, cover with slide and use microscope to measure diameter of randomly picked 50 cells from 10-15 different clumps. Use formula to calculate volume once you have radius length in hand. Volume of cells near the oocyte may be different from cells in periphery of follicle. Take an average. Good luck.

Hi Niloufar,

Hi Ahmed,

Thanks for your answers. Actually, miRNeasy-mini-kit could works good with tissues or high number of cells but it will not be enough for fine samples as 25 nucleolus/sample.

I found this protocol which could be useful :

Thanks again for your help.

Dear Marayam ,

It is good to guide me and explain me how is it work

Regards,

Usama

Dear Gabor, the following paper may help you:

Mechanical Stresses in Embryonic Tissues: Patterns, Morphogenetic Role, and Involvement in Regulatory Feedback

L.V.Beloussov, S.V.SavelievI, .NaumidiV. V.Novoselov

Dear Hemraj,

The morphology assessment of sperm cells obtained from the testicular tissue by means of microTESE procedure is limited to motility, contour of sperm head, tail length and mid piece in relation to presence/absence of cytoplasmic droplet only! Most of the Embryologists including me follow the similar criteria for selection of testicular sperms prior to inject oocytes to anticipate healthy fertilisation rate.

This is so because of practicality of the TESE-ICSI treatment option in relation to availability of the motile and viable sperms as many a time we are having a very few number of sperms for performing ICSI which restrict us to apply additional laboratory test for evaluation of the testicular Sperm morphology. Overall Immaturity (chemica/ cytoplasmic/ Nuclear) of the Sperm is one of the important aspect for the normal growth of quality embryos and subsequent success of such TESE-ICSI-ET cycles. I hope, you might be convinced by the answer. However, latest publication also suggest no significant benefit of ICSI in non-male factor ART cycle given as follows:

📷📷Article Navigation

Z Li A Y Wang M Bowman K HammarbergC Farquhar L Johnson N Safi E A SullivanHuman Reproduction, dey118, https://doi.org/10.1093/humrep/dey118Published: 12 June 2018 Article history

What is the cumulative live birth rate following ICSI cycles compared with IVF cycles for couples with non-male factor infertility?

ICSI resulted in a similar cumulative live birth rate compared with IVF for couples with non-male factor infertility.

The ICSI procedure was developed for couples with male factor infertility. There has been an increased use of ICSI regardless of the cause of infertility. Cycle-based statistics show that there is no difference in pregnancy rates between ICSI and IVF in couples with non-male factor infertility. However, evidence indicates that ICSI is associated with an increased risk of adverse perinatal outcomes.

A population-based cohort of 14 693 women, who had their first ever stimulated cycle with fertilization performed for at least one oocyte by either IVF or ICSI between July 2009 and June 2014 in Victoria, Australia was evaluated retrospectively. The pregnancy and birth outcomes following IVF or ICSI were recorded for the first oocyte retrieval (fresh stimulated cycle and associated thaw cycles) until 30 June 2016, or until a live birth was achieved, or until all embryos from the first oocyte retrieval had been used.

Demographic, treatment characteristics and resulting outcome data were obtained from the Victorian Assisted Reproductive Treatment Authority. Data items in the VARTA dataset were collected from all fertility clinics in Victoria. Women were grouped by whether they had undergone IVF or ICSI. The primary outcome was the cumulative live birth rate, which was defined as live deliveries (at least one live birth) per woman after the first oocyte retrieval. A discrete-time survival model was used to evaluate the cumulative live birth rate following IVF and ICSI. The adjustment was made for year of treatment in which fertilization occurred, the woman’s and male partner’s age at first stimulated cycle, parity and the number of oocytes retrieved in the first stimulated cycle.

A total of 4993 women undergoing IVF and 8470 women undergoing ICSI had 7980 and 13 092 embryo transfers, resulting in 1848 and 3046 live deliveries, respectively. About one-fifth of the women (19.0% of the IVF group versus 17.9% of the ICSI group) had three or more cycles during the study period. For couples who achieved a live delivery, the median time from oocyte retrieval to live delivery was 8.9 months in both IVF (range: 4.2–66.5) and ICSI group (range: 4.5–71.3) (P = 0.474). Fertilization rate per oocyte retrieval was higher in the IVF than in the ICSI group (59.8 versus 56.2%, P < 0.001). The overall cumulative live birth rate was 37.0% for IVF and 36.0% for ICSI. The overall likelihood of a live birth for women undergoing ICSI was not significantly different to that for women undergoing IVF (adjusted hazard ratio (AHR): 0.99, 95% CI: 0.92–1.06). For couples with a known cause of infertility, non-male factor infertility (female factor only or unexplained infertility) was reported for 64.0% in the IVF group and 36.8% in the ICSI group (P < 0.001). Among couples with non-male factor infertility, ICSI resulted in a similar cumulative live birth rate compared with IVF (AHR: 0.96, 95% CI: 0.85–1.10).

Data were not available on clinic-specific protocols and processes for IVF and ICSI and the potential impact of these technique aspects on clinical outcomes. The reported causes of infertility were based on the treating clinician’s classification which may vary between clinicians.

This population-based study found ICSI resulted in a lower fertilization rate per oocyte retrieved and a similar cumulative live birth rate compared to conventional IVF. These data suggest that ICSI offers no advantage over conventional IVF in terms of live birth rate for couples with non-male factor infertility.

No specific funding was received to undertake this study. There is no conflict of interest, except that M.B. is a shareholder in Genea Ltd.

N/A.

assisted reproduction, ICSI, IVF, IVF/ICSI outcome, infertility, non-male factor infertility, male factor infertility, cumulative rates, live birthIssue Section: Reproductive epidemiology

You need to have enough DNA for comfortable PCRing. It will be hit and trial. You may try with mouse oocytes after superovulation which are easy to get and if you are interested as a trial run. Buffalo oocytes will be much more difficult and expensive to pool for trial run. DNA of mouse and buffalo oocyte should be similar in quantity.

My other worry is contamination with granulosa cells, need several washings of oocytes to get rid of other cells,

More opinion from other experts can not be ruled out,

Best,

Subhash

You can get the map from the company U purchased the vector. Or may be ask the fellow who has given you.

Dear Hemraz,

Please have a look at this useful ResearchGate link and request the authors for feedback.

Good luck!

Interesting question.

check this link

Best wishes

Follow this link maybe helpful

regards

P-value is a statistical parameter to decide whether a difference between 2 measurements may have occurred by chance at some threshold level of certainty. It may be used to decide whether the effect observed is likely to be real, or just random variation. If the experiment was designed to find out whether something was an inhibitor, the P-value allows you to state the likelihood that the effect observed was not due to chance, i.e. that there really was inhibition. The P-value, however, is not a measurement of the potency of inhibition.

If the experiment being performed was an inhibition measurement (i.e. there were 2 substances involved: a transport substrate and a potential inhibitor of the transporter), then the statement "uptake less than 50%" (referring to the transport substrate's uptake) indicates that inhibition was >50%. Therefore, ignoring any scatter in the data, the IC50 of the inhibitor would be less than the concentration of the inhibitor used in the experiment. However, an IC50 should be calculated based on the effect of several concentrations of inhibitor, not just one.

It's definitely possible to knock-down lncRNA or miRNAs in the nucleus with antisense.

You may find this information helpful:

and this webinar:

I have no personal experience in doing this in mouse oocytes, but would suggest you look at microinjection of the antisense oligo.

Below is a publication from Ionis scientists, the experts in antisense technology - maybe you could try and contact the first author and see if they can help?

Unless you find multiple studies that made use of ketoconazole as antifungal for cell culture treatments, I advise you not to use it. Ketoconazole has been documented to antagonize GRs (glucocorticoid receptors) and if your aim is to maintain the integrity of the cells after an antifungal treatment, your drug of choice might not be appropriate.

Check if this supplement might be of lower price that Amphotericin: http://www.invivogen.com/fungin

You have to work under completely asceptic conditions and in Laminar Flow Cabinet containing filtered air. Sterilize your hands and sterilize the cabinet you work in .Also lab. atmosphere should be sterilized overnight with UV and switch off in the morning befor you enter to your Lab. Also you can use fungicides

Dear Islam,

I would recommend you to check the CO2 concentration in your incubator and the pH of your media. In general, somatic cells (like cumulus cells and fibroblasts) like to grow in slightly alkaline media (pH around 7.5 - 7.6). Maybe by just adjusting your culture conditions you can prevent them from growing so much and also your oocytes will like it better.

Tunel, although not 100% specific, is a good first step approach.

Best regards,

Daniela Rodler

To avoid what Anjul is mentioning, it is preferable to only do macropatches in Oocytes.

Here are some Refs: http://www.springerprotocols.com/cdp/search/searchResultPage?text=Macropatch

Hi Haonan, 

I am not a Xenopus- guy but rather a fly/zebrafish one. I hope it is still useful.

There are a plethora of potential problems:

- age of the parents (egg quality drops with age)

-toxicity of the injected agent or the buffer it is dissolved in

- check the pH and osmolarity of all your buffers 

 - filter sterilize buffers if appropriate

-we keep the larvae for 2 days in 1/100 PenStrep in E3 with methylene blue and check 3 times the first day for dead larvae. They start to decompose very fast and infect the remaining. The puncture wound is an entry point-for bacteria.

- use fewer larvae per dish to prevent infection

- did you use some RNAse-inhibitor in the injection mix? That could have deleterious effects. 

- make a control mock- injection without the RNA  check the parameters of the injecting apparatus. 

-check the parameters of the injecting apparatus

-monitor uninjected larvae in your buffer(s)

-do a literature search for the RNA you're injecting, is it plausible that it causes lethality? 

- check the lab's protocols if any change in water-quality occurred adults might tolerate some change better than larvae

-check your needles /puller- parameters are the needles of good quality in terms of length 

-did you use the right glass for them

-check if the MilliQ filter is OK

I hope this gives you a few ideas where to start.

Cheers 

David 

A.Retrovirus based iPSC reprogramming protocol

Retroviral reprogramming using standard Oct4, Sox2, c-Myc, and Klf4 Factors

iPSC colonies are selected based on morphology

AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)

5 vials iPSC colonies will be provided (>2 x 10^5 cells/vial)

Timelines for human iPSCs: 3-4 months and mouse iPSCs: 2-3 months

B. Episomal vector based iPSC reprogramming protocol

Episomal DNA reprogramming using Oct4, Sox2, c-Myc, and Klf4 factors with small molecules

iPSC colonies are selected based on morphology

AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)

5 vials iPSC colonies will be provided (>2 x 10^5 cells/vial)

Timelines for human iPSCs: 4-5 months

C. Protein based iPSC reprogramming protocol

Protein reprogramming using standard Oct4, Sox2, c-Myc, and Klf4 factors with a cell-penetrating peptide (CPP)

iPSC colonies are selected based on morphology

AP Staining and immunostaining of ESC markers (Nanog or Oct4, SSEA3 and TRA-1-81 included)

iPSC colonies will be provided (>5 x 10^5 cells/vial)

Timelines for human iPSCs: 3-4 months

You can find this information here http://www.creative-bioarray.com/Services/Custom-iPS-Cell-Services.htm

tnx a lot!

sorry for  answer back this late, there was a problem with my connection

regards, Sara Vahdat

Dear De Jesus. I think the following article is so useful for your request. Kind regards. https://www.ncbi.nlm.nih.gov/pubmed/22907499