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High Background level (in unstimulation well) of Rat IFN-g ELISPOT


Guest Yong-bok Seo

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Guest Yong-bok Seo

Hi Mabtech,

 

I have a trouble on my Rat IFN-g ELISPOT results (catalog number is 3220-4APW-10).

As you can easily assume, the background level of unstimulated well is too high to distinguish the spot number of stimulated well, even though I just performed the experiment as product's protocol.

Oddly, the backgroud was not observed when I perform Mouse IFN-g ELISPOT ((Becton Dickinson (BD) ELISPOT set)).

Althogh I had tried to apply BD protocols (Blocking & washing step) to reduce the backgroud level, it doesn't worked.

Please fine the attached detailed protocol of my Rat IFN-g ELISPOT following letter, and please give me the trouble-shooting way.

 

Sincerely,

 

Seo.

 

 

1. Materials.

(1) RPMI 1640 media (Hyclone, SH30027.01)

(2) FBS (fetal bovine serum) (Hyclone, SH30396.03)

(3) Antibiotics (Corning cellgro, 30-004-CI)

(4) R10 media (RPMI1640 + FBS (10%) + Antibiotics (1%))

(5) RBC lysis buffer (BD, 555899)

(6) PBS (Hyclone, SH30256.02)

(7) Tween20 (Merck, 8.17072.1000)

(8) Pure water (Hyclone, SH30538.01)

(9) wash buffer I (1X PBS containing 0.05% Tween 20)

(10) wash buffer II (1X PBS)

(11) Rat IFN-gamma ELISpot plus (APL) 10 plates (Mabtech, 3220-4APW-10)

(12) Concanavalin A (sigma-aldrich, C5275)

(13) Skim milk (BD, 232100)

(14) 15 mL conical tube (Falcon, 352096)

(15) 50 mL conical tube (Falcon, 352070)

(16) Disposable 5 mL pipette (Corning, CT-4487)

(17) Disposable 10 mL pipette (Corning, CT-4488)

(18) E-tube (Sorenson, 11510)

(19) 100mm dish (SPL, 10100)

(20) 150mm dish (SPL, 10150)

(21) Cell strainer (BD falcon, 352340)

(22) 5ml syringe (Koreavaccine, 150526)

(23) Cell counting tube

(24) peptide pool

 

2. Splenocytes preparation (in sterile condition)

(1) Place the spleen into a labeled conical tube containing 7ml of R10 media (RPMI1640 + FBS(10%) + Antibiotics (1%))

      (2) Place the spleen onto a 40um pore cell strainer attached onto a labeled 100mm dish.

(3) Press the excised spleen through the 40um pore cell strainer using the plunger end of a syringe.

(4) Wash the cells through the strainer with the excess 5ml of R10media.

(5) Centrifuge the cell suspension at 1,500 rpm for 5 minute at 4℃.

(6) Discard supernatant carefully, and vortex the cell pellet in 3 ml of 1X RBC lysis buffer.

(7) Incubate the cells for 5 minutes at room temperature.

(8) Stop reaction by adding 10ml of R10 media.

(9) Centrifuge the cell suspension at 1,500 rpm for 5 minute at 4℃

(10) Discard supernatant carefully, and resuspend the cells in 10ml of R10 media.

(11) Quick spin (just before 1500rpm) for debris down

(11) Transfer the cell (supernatant) into new labeled 15ml conical tube

(12) Centrifuge the cell suspension at 1,500 rpm for 5 minute at 4℃

(13) Discard supernatant carefully, and resuspend the cells in 10ml of R10 media.

(14) Prepare the counting tube, and add 450ul of R10 media in each tube. And transfer 50ul of cells into counting tube

(15) Counting the splenocyte set as lymphocyte cell type, 10-fold dilution.

(Vicell counter, Beckman coulter)

 

3.  ELISpot (in sterile condition, step (1)-(7))

 

3.1 Preparation of ELISpot plate

(1) Remove the plate from the sealed package and wash 4 times with sterile PBS (200ul/well)

(2) Add 200 μl/well of 5% skim milk in PBS and incubate for 1 hr at 37ºC, in a 5% CO2 incubator

 

3.2. Incubation of cells in plates

(3) Adjust cell (from step 2. Splenocytes preparation) concentrate at 5x10^6 cells/ml by removing or adding media.

          * The cells of ConA stimulant would be seed at 5x10^4 cells/well.

      (4) Preparation of stimulant mixture

      * Positive control : Concanavalin A (ConA), Dilute the ConA (1:1000) in R10 media.

(5) Discard blocking solution carefully and add stimulant mixture (prepared by step (5)) 100µl/well

  (6) Add 100ul of cells (prepared by step (4)) into each well.

(7) Incubate at 37℃, 5% CO2 incubator for over-night.

 

3.3. Detection of spots

(8) Remove the cells by emptying the plate and wash 5 times with Wash buffer II (1XPBS), 200ul/well

(9) Dilute the detection antibody (rFINγ-II-biotin) to 1ug/ml in PBS containing 0.5% FBS (PBS-0.5% FBS). Add 100ul/well and incubate 2hours at room temperature.

        (10) Wash plate as above (step (9))

(11) Dilute the Streaptavidin-ALP (1:1000) in PBS-0.5% FBS and add 100ul/well. Incubate for 1hour at room temperature.

       (12) Wash plate as above (step (9))

(13) Filter the ready-to-use substrate solution (BCIP/NBT-plus) through a 0.45um filter and add 100ul/well. Develop until distinct spots emerge (for 10-30min)

(14) Stop color development by washing extensively in tap water. If desirable, remove the under-drain (the soft plastic under the plate) and rinse the underside of the membrane.

     (15) air-dry

   (16) Inspect and count spots in an ELISpot reader or in a dissection microscope.

 

 

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Dear Seo,

 

 

Reading through your post there is nothing that gives me an obvious hint as to why you should have issues with high backgrounds. Well maybe one thing, you have blocked your plates using 5% skim milk in PBS. This is not part of the Mabtech protocol. We recommend using cell culture medium with FCS for 30 minutes blocking. If the milk solution/powder gets contaminated, high backgrounds could be the result. 

 

For me to give better feedback, it would be very helpful if you could post images of your elispot wells, one stimulated and one unstimulated well. This will help me see what kind of background issue you are experiencing. Is it pure artifacts or is it real spots? Is it a general darkening of the membrane or is the background in fact cytokine secreting cells?

 

 

best,

Christian

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  • 2 weeks later...
Guest Yong-bok Seo

Dear Christian

 

Thanks for your answer. There is one thing to correct on my experimental protocol. I have blocked the plates with not 5% skim milk in PBS but with R10 media (10% FBS in RPMI). Sorry for confusing you. and I couldn't find the way to post the image file of my ELISPOT data. I'll appreciate if you let me know the way to post my results images.

 

Best,

 

Seo.

   

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Dear Christian

 

Thanks for your answer. There is one thing to correct on my experimental protocol. I have blocked the plates with not 5% skim milk in PBS but with R10 media (10% FBS in RPMI). Sorry for confusing you. and I couldn't find the way to post the image file of my ELISPOT data. I'll appreciate if you let me know the way to post my results images.

 

Best,

 

Seo.

 

Dear Seo,

 

Ok, so you did use medium containing 10% FCS. That is a good thing!

 

In order for you to post images you have to register in our forum. Just hit the create account button in the right hand corner and follow the instructions. Once logged in you are able to attach most files, including JPEGs and Powerpoint files. I would prefer a powerpoint if  you have that prepared. Please take the orginal images from the reader that are stored in the saved folder. Do not give a print-screen as resolution is too low in most cases.  

 

After you upload I will be able to give you much more feedback.

 

/Christian 

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Guest Yong-bok Seo

Hey Seo,

 

Turns out I was wrong. You do not have to register in order to post images. I just needed to switch a setting inside my admin control panel. Please come back and upload :)

 

  Hey Christian,

 

I posted my experimental data file. Please check and comment about it.

 

Many thanks.

 

Seo.Mabtech_ELISpot_plate.pptx

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Dear Seo,

 

I have looked in your attached powerpoint and here are my comments:

 

1. The spots look technically very good. You have done the assay correctly. Plates are pre-coated so its easier of course but always good to see that the quality of spots are in fact like they should be. Cudos. 

 

2. In your attached powerpoint, the same slide is repeated 2 times. You show unstimulated and "peptides". It would be interesting to see how the ConA wells at 50,000 cells look as well.

 

3. There is a tendency for bigger spots in the peptide stimulated wells, indicating a "response". However, just like you say, the background from unstimulated cells is surprisingly high, even at 500,000 cells/well. This could be due to a number of reasons, for example:

 

- During the preparation of the spleen, cells have been activated by some contaminating factor. 

 

- During the setup of the plate, conA was accidentally put in the medium controls. If you first pipette ConA wells with stimuli, and then put pure medium into the other wells without changing the tips of the multichannel pipette, this could be the end result. Also, splashes of ConA could have ended up in the medium controls from surrounding wells, during plate setup. I have done these mistakes myself. 

 

- We know from experience that bad batches of fetal calf serum (FCS/FBS) can cause high backgrounds in IFN-g ELISpot. Are you using a new batch? Have you used the serum before for the earlier mouse IFN-g ELISpot experiments?

 

- It could be that the result you are seeing is totally correct. Cells have not been contamined with anything and there is nothing wrong with the experiment setup. Instead, the T-cells of the rat spleen did in fact secrete large amounts of IFN-g due to their in vivo history. Can you tell us something about these rats? Have they been exposed to something special, something that would perhaps make the T-cells in the spleen secrete IFN-g before they were taken out?

 

 

best,

Christian

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