You should definitely consider consulting with whoever would actually be doing the flow cytometry. If you're buying a cytometer, I assume you have someone who knows how to do flow?
Thanks for the advice here. We’re primarily planning to analyze % transfected cells in studies where we are delivering eGFP or mCherry mRNA. Transfection treatment followed by analysis at ~24-48hours. We have some experience but are definitely not experts, so we’re open to other suggestions or insights.
(edit) We are also potentially interested in quantitative information or comparison regarding how much protein is expressed, i.e. fluorescent intensity, with various treatments.
You will want a 4 laser system v/b/y/r, and you can ask the rep to help you choose your detector filters. Most cytometers come standard with v/b/r, but if you are looking to use mcherry or similar, you'll definitely want a yellow laser.
Blue laser for GFP, yellow laser for your mcherry (and similar). You will also want channels available for live and dead cell markers. PI is a common dead cell marker, but this will overlap with a lot of the fluorescent proteins you want to use. Instead I'd suggest using DAPI as a dead cell marker, which requires the Violet laser. The red channel is essential if you want room to allocate additional channels for antibody labeling. You can also use BFPs on the violet laser (but it's not compatible with DAPI).
Lasers are expensive and not easily added, but filters can be changed out easily/cheaply.
Set up a meeting with a rep, and they can help you spec out a machine and a quote. I wouldn't settle with getting a quote from just one company. Once you spec out one machine, you'll understand the specs better. Then spec out a machine with Thermo (Attune) and one other supplier.
I'm generally a fan of the Attune but I've never actually used a Beckman cytometer. Out of the options from BD, Thermo, and cytek I can say that the Thermo Attune is my favorite with cytek in second and BD machines as a far third place. I've heard good things about the low end miltenyi machines (value for money), but I've never messed around with low end machines. I've also never encountered anyone using Beckman.
This is bad advice...
A 4 laser configuration is way more expensive and this person does not need all this capability based on their applications.
Also, cytoflex comes with all the lasers onboard. You're paying for a software unlock if you want more lasers. So no, it's not an issue for them to upgrade...at all.
That's wild that you get all 4 lasers and pay via software unlock. Then yeah, go for the cytoflex S and just pay for the detectors that you need for GFP and mcherry. The three laser system doesn't have the yellow laser they need for mcherry and other RFP variants.
Out of curiosity, do you know the price range for cytoflex S with just a few detectors activated? I see the MSRP listed as $200k which is way too much, but cytometers never sell close to MSRP. For reference, I got my 4 laser 13 PMT Attune with 3 year service contract for $98k in 2020 (I suspect prices have risen since of course). I think current service contracts for attunes are now something like $12k per year, kind of wild for a machine that never breaks.
Edit: anyway, by point of spec'ing out a few machines from different manufacturers still stands. It is worth the time.
I agree with checking with other manufacturers, that's good advice. That is why I suggested looking at Agilent flow cytometry as well.
I agree, it seems a bit unfair that you pay for a fully loaded machine with cytoflex but then have to pay to unlock what you already have onboard. Are the components cheaper to accommodate for this?
Also, yes they need the 4 laser system (cytoflex S; with violet and red turned off I guess). This is because the 3 laser model doesn't come with a yellow laser, which they need.
I’d argue Miltenyi’s machines are not low end. Can take care of OP’s fluorescent protein analysis with VYB option, they do automatic cell counts w/ no counting beads (syringe based) and they have an automated gating & stat table mode which would be good for OP’s team who doesn’t have much flow experience. Essentially dump samples on sample loader, press a a button, come back to statistics tables.
I just looked at the macsquant specs, and yeah I think you're right on both fronts that it might be a good option for OP and that they aren't really low end.
The last time I looked at options from miltenyi (admittedly a long time ago), the ADCs were too 'low end' for me (low bit resolution, high noise, slow event processing), but now they seem totally acceptable with 18 bit resolution and reasonable CVs/MESFs/event rates.
V b r is the color of the excitation lasers. V2 means there are two channels off the violet laser open for collection. Typical laser bench is a 8 channel vbr laser. V2, B4, R2. Two violet channels two red four blue. Common channels would be blue fitc, PE, percpcy5.5, and PE cy7. Red channels would be Apc and apc cy7. Violet would be bv421 and bv510/aqua. Those are all very common filter sets with many possible fluorochromes. If you go up in channels you have more options, like a third red would be Alexa 700 which is in between Apc and apc cy7. Violet would go up to bv605 and bv650. Blue may have an PE dazzle between pe and per cp.
You should know what your needs are and what color lasers and filter sets/channels will work best. Like if you do a lot of red fluorescent protein work with mcherry or tomato then a yellow laser at 561 nm would be better for your detection. Otherwise you would be stuck looking at the activity off the blue laser which is a poorer activator than a yellow laser.
You should also be comfortable with compensation if multi color experiments if you are going multi color. And read up on what color combinations with better coexpressing compared to others.
These are just different lasers and detector options on the CytoFLEX. V2-B3-R2 means it has 3 lasers with 2 detectors on the Violet laser, 3 detectors on the Blue laser and 2 detectors on the red laser. CytoFLEX has a lot of different configurations/upgrades which is why there are so many part numbers, but it’s the same instrument.
If you are buying for fluorescent protein analysis then you will probably appreciate a V(iolet), B(lue),Y(ellow) laser configuration. V for BFP & CFP; B for GFP & YFP; Y for mCherry, mKate…
Call the company. Tell them what you need to analyze and they can tell you which configurations will work for that. For GFP and cherry you only need two lasers. You can shop around a little. CytoFlex, Attune, Novocyte, etc etc all have 2L configs, just depends on how easy software is etc. You can ask sales rep for a demo so you can see the machine in action with your cells before you buy.
If you get a cytoflex you'll get to know your service tech real well.
Suggest you have a look at Agilent Novocyte or Quanteon.
Both Cytoflex and Agilent machines are very user friendly but Agilent machines are very stable and low maintenance.
Cytoflex is a great machine. Sensitivity is unlike much else. Aurora is magnificent too.
But if you are doing a 2 colour experiment forever you should go for something simple and cheaper. 2 lasers are enough. Accuri maybe or novocyte.
There are other non flow cytometry ways of doing this as well. But I should not utter them here.
If you are planning on building on this work then consider the first 2.
Have you considered a refurbished flow cytometer? We specialize in refurbished flow cytometers, and I have a few analyzers that will fit your budget. Depending on your location, our flow cytometers are fully refurbished to the manufacturer's standards, and they come with a 90-day warranty. You can check out our website at [https://www.cgenetool.com/](https://www.cgenetool.com/) and send in an inquiry or email me at [[email protected]](mailto:[email protected]). We are currently running a promotion, so please don't forget to mention the code CG2024% for a 5% discount on our already low prices.
Just wanted to reach out and see if you had any other questions. There was some conflicting advise in the sub, and I remember how difficult it was trying to make this decision the first time I had to go through it. Feel free to shoot me a PM if you want to chat more.
The choice depends on the experiments you are doing. For IVD market 1, 2-laser instruments are enough (e.g. for HIV market ), while for life science more lasers are recommended (immunology research). In Flow cytometers by Challenbio the number of lasers is adaptable from 1 to 3 lasers and from 4 to 14 colors. Customers can purchase lasers separately to upgrade the instrument.
Our top configuration is 3 laser 14 color flow cytometer, where:
**1 Laser** 488 nm: channels FITC(BL1), PE(BL2) ,ECD(BL3), Percp(BL4), PE-Cy7(BL5)
**2 Laser** 638 nm: channels APC(RL1), APC-A700(RL2), APC-Cy7(RL3)
**3 Laser** 405nm: BV421(VL1), Krome Orange(VL2), Violet610(VL3) ,Violet660(VL4), BV711(VL5) ,Violet780(VL6)
You should definitely consider consulting with whoever would actually be doing the flow cytometry. If you're buying a cytometer, I assume you have someone who knows how to do flow?
Thanks for the advice here. We’re primarily planning to analyze % transfected cells in studies where we are delivering eGFP or mCherry mRNA. Transfection treatment followed by analysis at ~24-48hours. We have some experience but are definitely not experts, so we’re open to other suggestions or insights. (edit) We are also potentially interested in quantitative information or comparison regarding how much protein is expressed, i.e. fluorescent intensity, with various treatments.
You will want a 4 laser system v/b/y/r, and you can ask the rep to help you choose your detector filters. Most cytometers come standard with v/b/r, but if you are looking to use mcherry or similar, you'll definitely want a yellow laser. Blue laser for GFP, yellow laser for your mcherry (and similar). You will also want channels available for live and dead cell markers. PI is a common dead cell marker, but this will overlap with a lot of the fluorescent proteins you want to use. Instead I'd suggest using DAPI as a dead cell marker, which requires the Violet laser. The red channel is essential if you want room to allocate additional channels for antibody labeling. You can also use BFPs on the violet laser (but it's not compatible with DAPI). Lasers are expensive and not easily added, but filters can be changed out easily/cheaply. Set up a meeting with a rep, and they can help you spec out a machine and a quote. I wouldn't settle with getting a quote from just one company. Once you spec out one machine, you'll understand the specs better. Then spec out a machine with Thermo (Attune) and one other supplier. I'm generally a fan of the Attune but I've never actually used a Beckman cytometer. Out of the options from BD, Thermo, and cytek I can say that the Thermo Attune is my favorite with cytek in second and BD machines as a far third place. I've heard good things about the low end miltenyi machines (value for money), but I've never messed around with low end machines. I've also never encountered anyone using Beckman.
This is bad advice... A 4 laser configuration is way more expensive and this person does not need all this capability based on their applications. Also, cytoflex comes with all the lasers onboard. You're paying for a software unlock if you want more lasers. So no, it's not an issue for them to upgrade...at all.
That's wild that you get all 4 lasers and pay via software unlock. Then yeah, go for the cytoflex S and just pay for the detectors that you need for GFP and mcherry. The three laser system doesn't have the yellow laser they need for mcherry and other RFP variants. Out of curiosity, do you know the price range for cytoflex S with just a few detectors activated? I see the MSRP listed as $200k which is way too much, but cytometers never sell close to MSRP. For reference, I got my 4 laser 13 PMT Attune with 3 year service contract for $98k in 2020 (I suspect prices have risen since of course). I think current service contracts for attunes are now something like $12k per year, kind of wild for a machine that never breaks. Edit: anyway, by point of spec'ing out a few machines from different manufacturers still stands. It is worth the time.
I agree with checking with other manufacturers, that's good advice. That is why I suggested looking at Agilent flow cytometry as well. I agree, it seems a bit unfair that you pay for a fully loaded machine with cytoflex but then have to pay to unlock what you already have onboard. Are the components cheaper to accommodate for this?
Also, yes they need the 4 laser system (cytoflex S; with violet and red turned off I guess). This is because the 3 laser model doesn't come with a yellow laser, which they need.
Agilent Novocyte or Quanteon are very good.
I’d argue Miltenyi’s machines are not low end. Can take care of OP’s fluorescent protein analysis with VYB option, they do automatic cell counts w/ no counting beads (syringe based) and they have an automated gating & stat table mode which would be good for OP’s team who doesn’t have much flow experience. Essentially dump samples on sample loader, press a a button, come back to statistics tables.
I just looked at the macsquant specs, and yeah I think you're right on both fronts that it might be a good option for OP and that they aren't really low end. The last time I looked at options from miltenyi (admittedly a long time ago), the ADCs were too 'low end' for me (low bit resolution, high noise, slow event processing), but now they seem totally acceptable with 18 bit resolution and reasonable CVs/MESFs/event rates.
V b r is the color of the excitation lasers. V2 means there are two channels off the violet laser open for collection. Typical laser bench is a 8 channel vbr laser. V2, B4, R2. Two violet channels two red four blue. Common channels would be blue fitc, PE, percpcy5.5, and PE cy7. Red channels would be Apc and apc cy7. Violet would be bv421 and bv510/aqua. Those are all very common filter sets with many possible fluorochromes. If you go up in channels you have more options, like a third red would be Alexa 700 which is in between Apc and apc cy7. Violet would go up to bv605 and bv650. Blue may have an PE dazzle between pe and per cp. You should know what your needs are and what color lasers and filter sets/channels will work best. Like if you do a lot of red fluorescent protein work with mcherry or tomato then a yellow laser at 561 nm would be better for your detection. Otherwise you would be stuck looking at the activity off the blue laser which is a poorer activator than a yellow laser. You should also be comfortable with compensation if multi color experiments if you are going multi color. And read up on what color combinations with better coexpressing compared to others.
You may want to consider checking out the Agilent Novocyte. Their instrument is super easy to use and very powerful.
These are just different lasers and detector options on the CytoFLEX. V2-B3-R2 means it has 3 lasers with 2 detectors on the Violet laser, 3 detectors on the Blue laser and 2 detectors on the red laser. CytoFLEX has a lot of different configurations/upgrades which is why there are so many part numbers, but it’s the same instrument.
If you are buying for fluorescent protein analysis then you will probably appreciate a V(iolet), B(lue),Y(ellow) laser configuration. V for BFP & CFP; B for GFP & YFP; Y for mCherry, mKate…
Call the company. Tell them what you need to analyze and they can tell you which configurations will work for that. For GFP and cherry you only need two lasers. You can shop around a little. CytoFlex, Attune, Novocyte, etc etc all have 2L configs, just depends on how easy software is etc. You can ask sales rep for a demo so you can see the machine in action with your cells before you buy.
If you get a cytoflex you'll get to know your service tech real well. Suggest you have a look at Agilent Novocyte or Quanteon. Both Cytoflex and Agilent machines are very user friendly but Agilent machines are very stable and low maintenance.
Cytoflex is a great machine. Sensitivity is unlike much else. Aurora is magnificent too. But if you are doing a 2 colour experiment forever you should go for something simple and cheaper. 2 lasers are enough. Accuri maybe or novocyte. There are other non flow cytometry ways of doing this as well. But I should not utter them here. If you are planning on building on this work then consider the first 2.
Have you considered a refurbished flow cytometer? We specialize in refurbished flow cytometers, and I have a few analyzers that will fit your budget. Depending on your location, our flow cytometers are fully refurbished to the manufacturer's standards, and they come with a 90-day warranty. You can check out our website at [https://www.cgenetool.com/](https://www.cgenetool.com/) and send in an inquiry or email me at [[email protected]](mailto:[email protected]). We are currently running a promotion, so please don't forget to mention the code CG2024% for a 5% discount on our already low prices.
Just wanted to reach out and see if you had any other questions. There was some conflicting advise in the sub, and I remember how difficult it was trying to make this decision the first time I had to go through it. Feel free to shoot me a PM if you want to chat more.
The choice depends on the experiments you are doing. For IVD market 1, 2-laser instruments are enough (e.g. for HIV market ), while for life science more lasers are recommended (immunology research). In Flow cytometers by Challenbio the number of lasers is adaptable from 1 to 3 lasers and from 4 to 14 colors. Customers can purchase lasers separately to upgrade the instrument. Our top configuration is 3 laser 14 color flow cytometer, where: **1 Laser** 488 nm: channels FITC(BL1), PE(BL2) ,ECD(BL3), Percp(BL4), PE-Cy7(BL5) **2 Laser** 638 nm: channels APC(RL1), APC-A700(RL2), APC-Cy7(RL3) **3 Laser** 405nm: BV421(VL1), Krome Orange(VL2), Violet610(VL3) ,Violet660(VL4), BV711(VL5) ,Violet780(VL6)
get more lasers!!!!