May I ask po Doc how did you do the quantification? How did you count the blood vessels? Can it be just done using a microscope or is there a software needed? Any suggestions po? Thank you po.
I have a question po Doc. Do you administer the treatments the same day that you opened the eggs? And where did you inject the treatments po, in the blastodisc po ba or allantois?
hello po doc! I hope you you respond to this comment. I would really like to ask po what software your team used in quantification of the vessels. Was it done manually and how do you verify it if it was through manual method po? 😭
Thank you for posting the video, we are watching it multiple times to prepare for doing a CAM assay ourselves. For your needle prick, did you make that by putting a needle inside of a rubber stopper? Do you by chance know the size of the needle you used for the end of the egg vs where the blood vessels are? Do you use the Teflon ring when implanting tumor cells?
We didn’t use Teflon tape. But we used parafilm or sterilized tapes to seal the eggs. For the needle sizes, I am not really sure about it. The previous scientists in our lab made those. Maybe as long as it is enough to puncture tiny holes onto the egg shells, then it’s good. Maybe use 23 gauge and 14 gauge (?)
Hello! We determined the anti-tumor effects of our treatments by measuring the weight of the implanted tumors after days of exposure to the treatment. For the antiangiogenic effects, we measured the hemoglobin level.
Hello po!!! Ang insightful po talaga ng videos niyo, magtatanong po sana ako kung paano niyo po yung disposal niyo nung eggs and if may literature po kayong ginamit as basis?
Hi! You don't really need to have a literature for the disposal. haha As long as you have a biopit or your institution has good safe handling and disposal of laboratory animal waste, then you're good to go.
Hi! We did not aspirate the albumin on this protocol. Instead, we displaced the airsac from it’s original position to the top of the CAM. Aspirating the albumin or doing our step will just move the CAM a little downward to give us enough space when during treatment and observation.
@@DoctorJPTV Thank you so much! I have a few more questions. What diameter for the window do you recommend best? I’m working on an in ovo CAM assay and we stated a 1x1 cm window, but other studies suggested a 3 cm diameter. If we use 3 cm, will that affect the sterility? And will a parafilm paper along suffice or sealing? Sorry for these 😭
@@lolypick the window size really depends on the how you will treat your CAM. If you’ll just insert a filter paper or gel inside, then drill or cut a hole with enough size so you can insert those materials, 1x1cm is enough for that. Also, smaller window size helps prevent contamination and is easier to seal with tape or parafilm.
Hi.. Sir have you perform research about anti-angiogenic effect on check embryo using CAM.. If ever what is the bases that you can say that your research is successful. Thank you in advance
Hello! If you want to determine the anti-angiogenic effect of your treatment, you can check the proliferation of new blood vessels. You can either count the number of branching points from the primary blood vessel to its tertiary or you can also measure the hemoglobin level. Angiogenesis is the formation of new blood vessels. So, you want to inhibit this process. You know you have inhibited this process, when you have 1. Small number of new blood vessels 2. Lower level of hemoglobin These are just the basic parameters. You can look for more by reading RRLs related to CAM Assay.
@@DoctorJPTVHello po Doc, 4th year BS Biology student here at kasalukuyang nagka CAM Assay. Ask ko lang po if ano ang ginamit niyong scoring and ang ginamit lang namin is filter paper disks, necessarily ba na sa loob ng disk or sa labas nun mag quantify ng mga branching points at blood vessels?
@@MhelvinRizal Hello! In this CAM assay (video) we didn’t use any filter paper disks and we didn’t quantify the branching points of blood vessels. If you used filter disks to introduce your treatments you should place it on top of the CAM or where the primary blood vessel is located. When quantifying the blood vessel, you should start counting from the 2nd branching points to the 3rd and 4th and soon (depending on your preference). The primary branching point or the Y serves as your guide where to start.
@@DoctorJPTV Hello doc, if we use filter paper disk, saan po ilalagay ung fikter paper? on top po ba ng Y blood vessel or beside? Like what is you criteria on where to put the treatment?
Sir sa CAM Assay need jud ang cancer cells? Naa man jui uban procedure sa uban mga study wla...like angiogenesis lang og morphometric ang gi na administer.
Hello po. We are currently conducting a CAM Assay about a plant's anti-angiogenic effect and all of our eggs died po. How can we make them survive for at least a day para lang maobserve namin yung blood vessels nila and macount po? And I have learned from your reply here sa comment section about the hemoglobin test level, paano po yon?
You should monitor all conditions properly, the humidity, temperature, etc. Measuring the Hemoglobin level is an alternative way of quantifying the angiogenesis. If mataas ang hemoglobin level, pro-angiogenesis. If mababa, anti-angiogenesis. But this is strictly for CAM assay with tumor implants. If walang tumor implant, you can use the commonly used technique, which is counting the 1st, 2nd, and 3rd branching points of the blood vessels.
hi po! what does the CAM assay usually help in identifying po? when it comes to angiogenesis po ba/determining anti-angiogenic effects, is it recommended to put tumor before the treatment, or just directly put the treatment. what difference will it make po, if so? also po sir what kind of tumors po yung iniimplant? sorry for the questions. it’s just that I have no one to consult about this. hope you see this and have a great day ahead.
Hello, the experimental setup depends on the goals of your research project. In this specific research design, our goal is to determine the anticancer effects of our treatments, specifically antiangiogenesis and antitumor effects. We implanted tumors (skin, pancreatic, and gastric tumors) because, again, we wanted to determine the antitumor effects of our treatment. If implantation of tumor is not feasible at your own laboratory, you can just determine the pro-angiogenic or antiangiogenic properties of your treatment by counting the number of blood vessels formed after being treated with your treatment. Example: if it formed more blood vessels compared with the untreated group, it means your treatment has PRO-angiogenic properties. If it formed lesser blood vessels or it prevented the formation of new blood vessels, it means your treatment has ANTIangiogenic properties.
@@DoctorJPTV i see! it seems na napaghalo ko po yung pagdetermine niyo ng anti-tumor and anti-angiogenic properties niyo po. now i understand, thank you so much!
@@DoctorJPTV huhu hi po ulit, kapag maglalagay na po ba ng extract need po ba iinject sa egg if oo sa may yolk po ba or idrop lang yung extract? And do we have to remove/peel ung sac after maalis ung shell?
Hi doc, kanang anova man jud among gamit sa statistical tool. Paano ba mi maka come up sa results nya e compute gamit anova? bilangon ba ang blood vessels after butang sa treatment? unta ma notice dooooc next week na among defensseeee!! Thankyou po.
Depending on your selected parameter. Sa amuang study kay hemoglobin level ang basehan sa angiogenic properties (anti ba or pro). In your case, if number of blood vessels ang inyong parameter, then need mo mag-count ug blood vessels (primary to 3rd branching points; search sa google for guide). Dapat naa mo replicates. Mao inyong gamiton for anova.
Hi! It really depends on the laboratory. I haven’t tried na nagpatest ng CAM. Kami mismo ang nagpeperform ng assay. As long as you have the right tools, it’s pretty simple.
@@bsit-johnmaridinglasan2787 Hi! For this specific test na nasa video, we used this pubmed.ncbi.nlm.nih.gov/24769074/. But you can look for a much easier and cheaper CAM assay online www.hvm.bioflux.com.ro/docs/2016.10-16.pdf.
hi po, our study po is to identify the anti angiogenic effect of crowfoot grass in CAM Assay, can we ask po if ilang days po ang duck embryo na ginamit niyo po?
Hi! In our experiment, we purchase the eggs from a facility that sells laboratory grade animal models. So, we bought 1-day old eggs and incubate it in our lab. If you're planning to purchase your eggs from a poultry or hatchery and you want to save time and budget (you pay for the incubator per day in your lab, I assume), you should make sure to buy fertile eggs. 4-day old eggs is a safe age to use for CAM Assay.
@@arasal7338 incubate it for 4 days more. After that, do your treatment then incubate it for 8 days. Then do your examination after that. That is how we did it in our experiment. You can modify this if you want, especially if you just want to quantify the number of blood vessel branching points.
Usually, 3-day old eggs are used for this quantification. Then 1 day incubation (acclimatization stage). Then treatment day. Then incubate for 4-7 days. Then examination.
@@DoctorJPTV Hi po ulit sir. Sir, we already conducted our study po about anti angiogenic activity of our plant extract using CAM assay po, but I only saw today sa reply niyo sa isang question po dito na you implanted tumors sa CAM bago niyo nilagay yung treatment niyo. Sa amin sir, wala na po namin nalagyan ng tumor ang cam, diretso na po lagay nung treatment, and that might resulted to our duck eggs to be dead po (3/9 lang po buhay). is there any way we can still count and quantify the branching points of the dead cam assay po??
We did this po and nagkamolds ang mabaho po yung eggs tapos nawala rin po yung ugat? Ano po yung possible na mali namin, and naka DIY incubator po kami
We're conducting a study po na similar sa angiogenesis, mag tuturok kami ng extract and to see if liliit/hinder ung growth ng veins and need po bilangin.
Contaminated incubator, not properly cleaned, not properly closed, not airtight door, etc. Better clean your incubator first before using. Maintain 37C temperature and 55% relative humidity.
@@DoctorJPTV Kapag nag-grow na po yung tumor pwede po ba lagyan ng drug or plant extract para ma-test po kung effective anti-cancer po sila? Thank you po.
Hi! Thank you for noticing. Yeah, I admit we should be wearing face masks to make the whole setup clean and to avoid contamination. But, yeah, our supervisor has been performing this assay a lot of times. And during this experiment, we contamination didn’t occur. 😊
Wow grabe this is amazing!!
Hello po, thank you for this wonderful demonstration mabuhay po kayo I was able to understand by watching thank you po
Wow naman. Ganito pala yun
ILOVEYOU! (Cheret!)
Love the voice over hah. 👏🏻
Jeffrey Lagrosa hahaha kalimot ko’g ingon sa camera 😂
First time ko maka kita ani. Interesting kaayo tanawon. 😍
omsimnida!
Hi, my study is to determine the Anticancer activity with belle pepper.
Can this method be use?
And what is the use for the matrigel please
Can you explain me how could I measure the effect after treatment? did you take photos? or which tecnique did you use?
hello sir, we are also conducting a research regarding CAM Assay, I would like to ask if what is the incubator used for the incubation of eggs?
Astig hahaha
May I ask po Doc how did you do the quantification? How did you count the blood vessels? Can it be just done using a microscope or is there a software needed? Any suggestions po? Thank you po.
May i ask po if anong scoring system ang ginamit niyo? Thanks po!
Hello sana po mareplyan. Ask ko lang po anong gamit niyong incubator sa laboratory and ano pong number ng temperature for cam assay. Thank you
paano buksan yung egg sir para maka pic for angiosys
I have a question po Doc. Do you administer the treatments the same day that you opened the eggs? And where did you inject the treatments po, in the blastodisc po ba or allantois?
Hello po! Do you know any laboratories here in the Philippines na nagpeperform ng CAM assay? Thank you po
hello po doc! I hope you you respond to this comment. I would really like to ask po what software your team used in quantification of the vessels. Was it done manually and how do you verify it if it was through manual method po? 😭
Thank you for posting the video, we are watching it multiple times to prepare for doing a CAM assay ourselves. For your needle prick, did you make that by putting a needle inside of a rubber stopper? Do you by chance know the size of the needle you used for the end of the egg vs where the blood vessels are? Do you use the Teflon ring when implanting tumor cells?
We didn’t use Teflon tape. But we used parafilm or sterilized tapes to seal the eggs.
For the needle sizes, I am not really sure about it. The previous scientists in our lab made those. Maybe as long as it is enough to puncture tiny holes onto the egg shells, then it’s good.
Maybe use 23 gauge and 14 gauge (?)
@@DoctorJPTV Thank you for your quick reply!
What is the mixture that you guys pouring in to the eggs
It's a mixture of matrigel and cancer cells.
Hello Can I ask a question??? What did you do to prove that your study has an effect or not??
Hello! We determined the anti-tumor effects of our treatments by measuring the weight of the implanted tumors after days of exposure to the treatment. For the antiangiogenic effects, we measured the hemoglobin level.
@@DoctorJPTV And How did you displaced the airsac?
@@LouiseDD. you can watch it starting at 0:44
Hello po!!! Ang insightful po talaga ng videos niyo, magtatanong po sana ako kung paano niyo po yung disposal niyo nung eggs and if may literature po kayong ginamit as basis?
Hi! You don't really need to have a literature for the disposal. haha As long as you have a biopit or your institution has good safe handling and disposal of laboratory animal waste, then you're good to go.
@@DoctorJPTV thank you po doc!!
Hello, we are currently working on CAM Assay. May I asked how are we going to ethically terminate our experiment po, like the disposal process?
Hi! We are currently working on CAM Assay as well. How did you ethically terminate your eggs po? TIA.
Hi! may I ask, you did not aspirate the albumin diba? if you are familiar with this, why is it employed on some procedures?
Hi! We did not aspirate the albumin on this protocol. Instead, we displaced the airsac from it’s original position to the top of the CAM. Aspirating the albumin or doing our step will just move the CAM a little downward to give us enough space when during treatment and observation.
@@DoctorJPTV Thank you so much! I have a few more questions. What diameter for the window do you recommend best? I’m working on an in ovo CAM assay and we stated a 1x1 cm window, but other studies suggested a 3 cm diameter. If we use 3 cm, will that affect the sterility? And will a parafilm paper along suffice or sealing? Sorry for these 😭
@@lolypick the window size really depends on the how you will treat your CAM. If you’ll just insert a filter paper or gel inside, then drill or cut a hole with enough size so you can insert those materials, 1x1cm is enough for that. Also, smaller window size helps prevent contamination and is easier to seal with tape or parafilm.
@@DoctorJPTV Thank you so much, doc! 😁 big help talaga. God bless po!
Hi. may i ask the reason for aspirating albumin?
Hi.. Sir have you perform research about anti-angiogenic effect on check embryo using CAM..
If ever what is the bases that you can say that your research is successful.
Thank you in advance
Hello! If you want to determine the anti-angiogenic effect of your treatment, you can check the proliferation of new blood vessels. You can either count the number of branching points from the primary blood vessel to its tertiary or you can also measure the hemoglobin level.
Angiogenesis is the formation of new blood vessels. So, you want to inhibit this process. You know you have inhibited this process, when you have
1. Small number of new blood vessels
2. Lower level of hemoglobin
These are just the basic parameters. You can look for more by reading RRLs related to CAM Assay.
@@DoctorJPTVHello po Doc, 4th year BS Biology student here at kasalukuyang nagka CAM Assay. Ask ko lang po if ano ang ginamit niyong scoring and ang ginamit lang namin is filter paper disks, necessarily ba na sa loob ng disk or sa labas nun mag quantify ng mga branching points at blood vessels?
@@MhelvinRizal Hello! In this CAM assay (video) we didn’t use any filter paper disks and we didn’t quantify the branching points of blood vessels.
If you used filter disks to introduce your treatments you should place it on top of the CAM or where the primary blood vessel is located. When quantifying the blood vessel, you should start counting from the 2nd branching points to the 3rd and 4th and soon (depending on your preference). The primary branching point or the Y serves as your guide where to start.
@@DoctorJPTV Hello doc, if we use filter paper disk, saan po ilalagay ung fikter paper? on top po ba ng Y blood vessel or beside? Like what is you criteria on where to put the treatment?
May I ask po doc if possible if paper disc would be used to apply the treatment instead of pippeting?
Apply where?
Sir sa CAM Assay need jud ang cancer cells? Naa man jui uban procedure sa uban mga study wla...like angiogenesis lang og morphometric ang gi na administer.
Nope. Di required ang cancer cells. Yeap, you can perform morphological analysis.
Hello po. We are currently conducting a CAM Assay about a plant's anti-angiogenic effect and all of our eggs died po. How can we make them survive for at least a day para lang maobserve namin yung blood vessels nila and macount po? And I have learned from your reply here sa comment section about the hemoglobin test level, paano po yon?
You should monitor all conditions properly, the humidity, temperature, etc.
Measuring the Hemoglobin level is an alternative way of quantifying the angiogenesis. If mataas ang hemoglobin level, pro-angiogenesis. If mababa, anti-angiogenesis. But this is strictly for CAM assay with tumor implants. If walang tumor implant, you can use the commonly used technique, which is counting the 1st, 2nd, and 3rd branching points of the blood vessels.
@@DoctorJPTV thank u po.
hi po! what does the CAM assay usually help in identifying po?
when it comes to angiogenesis po ba/determining anti-angiogenic effects, is it recommended to put tumor before the treatment, or just directly put the treatment. what difference will it make po, if so?
also po sir what kind of tumors po yung iniimplant?
sorry for the questions. it’s just that I have no one to consult about this. hope you see this and have a great day ahead.
Hello, the experimental setup depends on the goals of your research project.
In this specific research design, our goal is to determine the anticancer effects of our treatments, specifically antiangiogenesis and antitumor effects.
We implanted tumors (skin, pancreatic, and gastric tumors) because, again, we wanted to determine the antitumor effects of our treatment.
If implantation of tumor is not feasible at your own laboratory, you can just determine the pro-angiogenic or antiangiogenic properties of your treatment by counting the number of blood vessels formed after being treated with your treatment.
Example: if it formed more blood vessels compared with the untreated group, it means your treatment has PRO-angiogenic properties.
If it formed lesser blood vessels or it prevented the formation of new blood vessels, it means your treatment has ANTIangiogenic properties.
@@DoctorJPTV i see! it seems na napaghalo ko po yung pagdetermine niyo ng anti-tumor and anti-angiogenic properties niyo po. now i understand, thank you so much!
@@frankiestein615 in this design, we correlated the effects of our treatment on angiogenesis and tumor growth.
Can you give me a link to the material and methods used in this assay please
Hi there! Yeah, sure. You can use this for reference.
pubmed.ncbi.nlm.nih.gov/24769074/
ka laysho sa lab doc ang amoa kay nag use mi og 7 day old duck egg tas nag amend mi og solutions with different concentrations of a certain drug
Mao sad na among gihimo tung college 🥲
@@DoctorJPTV yes doc very devbio moments HAHAHAHA
Sir how much dosage ang ginamit nyo point every egg?
Hello! For this specific experiment, we 80 uL of 10 mg per plant extract and 30 ug per bioactive compound. These dose depends on your treatments.
@@DoctorJPTV what do you mean po, na et depends sa treatment po?
May I ask paano po ma displace yung aircell? Need po ba talaga yan to create a window? Or pwede po kahit dun na sa original spot?
Nasa video kung pano magdisplace ng aircell. Yes need gumawa ng window. Pwede naman original spot but might affect the result of your experiment.
@@DoctorJPTV huhu hi po ulit, kapag maglalagay na po ba ng extract need po ba iinject sa egg if oo sa may yolk po ba or idrop lang yung extract? And do we have to remove/peel ung sac after maalis ung shell?
Hi doc, kanang anova man jud among gamit sa statistical tool. Paano ba mi maka come up sa results nya e compute gamit anova? bilangon ba ang blood vessels after butang sa treatment? unta ma notice dooooc next week na among defensseeee!! Thankyou po.
Depending on your selected parameter. Sa amuang study kay hemoglobin level ang basehan sa angiogenic properties (anti ba or pro). In your case, if number of blood vessels ang inyong parameter, then need mo mag-count ug blood vessels (primary to 3rd branching points; search sa google for guide). Dapat naa mo replicates. Mao inyong gamiton for anova.
@@DoctorJPTV Thankyou po, ga libog sad mi sir unsa pag kuha sa positive og negative
@@dheveyanneberdon2337 positive and negative?
Hi po! Is there any way po we can contact your professor?
I can answer question. Feel free to send them to jpst.research@gmail.com 😊
Hi. Ask ko lang po sana kung pwedeng malaman kung magkano ang magpatest ng CAM Assay? Research purposes lang po sana, thank you! :)
Hi! It really depends on the laboratory. I haven’t tried na nagpatest ng CAM. Kami mismo ang nagpeperform ng assay. As long as you have the right tools, it’s pretty simple.
@@DoctorJPTV Ano po ang mga protocols na ginamit po para po sa CAM assay?
@@bsit-johnmaridinglasan2787 Hi! For this specific test na nasa video, we used this pubmed.ncbi.nlm.nih.gov/24769074/. But you can look for a much easier and cheaper CAM assay online www.hvm.bioflux.com.ro/docs/2016.10-16.pdf.
hi po, our study po is to identify the anti angiogenic effect of crowfoot grass in CAM Assay, can we ask po if ilang days po ang duck embryo na ginamit niyo po?
Hi! In our experiment, we purchase the eggs from a facility that sells laboratory grade animal models. So, we bought 1-day old eggs and incubate it in our lab. If you're planning to purchase your eggs from a poultry or hatchery and you want to save time and budget (you pay for the incubator per day in your lab, I assume), you should make sure to buy fertile eggs. 4-day old eggs is a safe age to use for CAM Assay.
@@DoctorJPTV oh okay po💖 but if we use a 4 day old egg embryo po, how many dayspo should we incubate the eggs po??
@@arasal7338 incubate it for 4 days more. After that, do your treatment then incubate it for 8 days. Then do your examination after that. That is how we did it in our experiment. You can modify this if you want, especially if you just want to quantify the number of blood vessel branching points.
Usually, 3-day old eggs are used for this quantification. Then 1 day incubation (acclimatization stage). Then treatment day. Then incubate for 4-7 days. Then examination.
@@DoctorJPTV Hi po ulit sir. Sir, we already conducted our study po about anti angiogenic activity of our plant extract using CAM assay po, but I only saw today sa reply niyo sa isang question po dito na you implanted tumors sa CAM bago niyo nilagay yung treatment niyo. Sa amin sir, wala na po namin nalagyan ng tumor ang cam, diretso na po lagay nung treatment, and that might resulted to our duck eggs to be dead po (3/9 lang po buhay). is there any way we can still count and quantify the branching points of the dead cam assay po??
Ask lang po if may specific na cancer po kayo na ginamit? At san po makakuha or makabili ng cancer cell?
Hello! For this experiment, we have implanted melanoma (A-375), gastric (AGS), and pancreatic (SUIT-2) cancer cells.
You can purchase cancer cell lines from suppliers/distributors like ATCC, Accegen, Thermofisher, etc.
Thank youupoo
We did this po and nagkamolds ang mabaho po yung eggs tapos nawala rin po yung ugat? Ano po yung possible na mali namin, and naka DIY incubator po kami
We're conducting a study po na similar sa angiogenesis, mag tuturok kami ng extract and to see if liliit/hinder ung growth ng veins and need po bilangin.
Contaminated incubator, not properly cleaned, not properly closed, not airtight door, etc. Better clean your incubator first before using. Maintain 37C temperature and 55% relative humidity.
@@DoctorJPTV we injected an extract and naka molds po, possible po ba na sa extract yun or sa incubator po mismo?
@@duzonaleeahalessandrag.934 incubator or you didn’t clean your eggs properly
@@DoctorJPTV actually our eggs has dirt po, pero sabi wag daw huhugasan, how do we clean it po ba without damaging the egg?
Pwede hingin ko po reference niyo po?
Hi there! Yeah, sure. You can use this for reference.
pubmed.ncbi.nlm.nih.gov/24769074/
@@DoctorJPTV Thank you po!
@@DoctorJPTV Hello ask lang po ako ulit, itong method na to makakagawa po to ng tumor po?
@@nicolebagas2322 Hi! For this specific test na ginawa namin, Yes. We implanted the tumors inside the eggs by mixing cancer cells in matrigel.
@@DoctorJPTV Kapag nag-grow na po yung tumor pwede po ba lagyan ng drug or plant extract para ma-test po kung effective anti-cancer po sila? Thank you po.
Why are the scientists not using face masks?
Hi! Thank you for noticing. Yeah, I admit we should be wearing face masks to make the whole setup clean and to avoid contamination. But, yeah, our supervisor has been performing this assay a lot of times. And during this experiment, we contamination didn’t occur. 😊
@@DoctorJPTV Thank you for sharing this nice technique.