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sample loop length on CyTOF1

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salong

Participant

Posts: 6

Joined: Thu Jun 11, 2015 2:47 pm

Post Thu Aug 20, 2015 7:38 pm

sample loop length on CyTOF1

We thought we had a 500 uL sample loop on our CyTOF1 - and may, but we'd like to confirm.

During a recent visit by a Fluidigm service engineer, we were told that it looked like we had a shorter sample loop. Fluidigm has sent us new 500uL sample loop to compare to ours (see picture) and our old one is shorter, but is this due to dead vlm in assembly (see second pix)?

It would be great if other CyTOF1 users could comment or share pix of your sample loop set-up and add any trouble-shooting you have done to set/determine sample loop length.

We do collect quite slowly and would like to get the most of our samples - we are looking for rare cells so every event counts!

Thanks

20150819_133910 (00000003).jpg
comparison

20150819_134710.jpg
set-up
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Aug 20, 2015 8:06 pm

Re: sample loop length on CyTOF1

Hi Alice,

I'm unclear on what you mean by "dead vlm in assembly": if each "loop" is connected to the same screw fittings (square fittings in your picture), then the dead volumes would be the same for each loop. By that, I mean the length of tubing downstream from your sample loop to the nebulizer capillary remains attached (and therefore the same), regardless of whether you have the old tubing or new tubing in place.

Unfortunately, I can't tell from the picture whether the internal diameters of the two lengths of tubing are different; therefore tubing length, in and of itself, isn't conclusive about whether your old tubing was 500uL, or less.

Ways to check:
1. I don't know what Luer lock fittings and such you might have on hand, from HPLCs/FPLCs and such. There are screw-to-syringe couplers, such as the one designated in orange on the attached PDF. If you can find something like that, then just empty the tubing, screw the fitting into the square connector. Fill a green syringe with water, attach it to the syringe end of the connector, and see how much volume it takes before the tubing starts dripping out the other end.

2. Since your CyTOFv1 runs at 45ul/min, attach one of the pieces of tubing to the machine. Load 500uL of sample (could be cells or beads), then hit run. Acquire for 10-12min (600-720 sec). Then repeat for the other piece of tubing. Then look at the two files in FlowJo or Cytobank, to see if one of them starts decreasing noticeably earlier than the other one.


Mike
Attachments
fittings.pdf
(340.61 KiB) Downloaded 316 times
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salong

Participant

Posts: 6

Joined: Thu Jun 11, 2015 2:47 pm

Post Tue Sep 01, 2015 3:32 pm

Re: sample loop length on CyTOF1

Thanks Mike, I wanted to share with others our troubleshooting and conclusions.

We decided to check the vlm's of our sample tubes by following Mike's suggestion #2 with beads. Attached is a pdf of our data for others to see. The top 4 plots are the 500 uL loop with injected volumes of 500 - 650 uL. The bottom 7 plots are the old loop with injected volumes of 250 - 500 uL.

Below are Mike's observations and suggestions based on this data. Thanks - we're feeling far more confident that we're acquiring all of our sample now and have the "right" sized sample loop!

----------------------------------------------
Your Old Loop plots definitely have their "hotspot" finish by ~300 seconds. By 400uL, your hotspot reaches 300 seconds and doesn't increase further even at 500uL. So, I would say that your Old Loop has a maximum capacity of *no more than* 400uL).

Your New Loop "hotspot" doesn't really increase past 400 sec, no matter your volume. Therefore, I would say its volume is 500uL.

Personally, I would go with New Loop, 500uL volume, 500 sec between injections, with 100-200 sec tail off at the end to minimize carryover without *having* to run Wash all the time.

1 Loop= 500 + 100 = 600sec
2 Loop= 500 + 500 + 200 = 1200 sec (could maybe get away with 1150 sec) etc.
Attachments
Loop Comparison.pdf
(159.25 KiB) Downloaded 308 times

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