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.
