TITLE: Fluorospot Assay
AUTHORS: cecilia , Alessandro Sette

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the steps required to collect and assay mouse anti-human cells using fluorospot assay.</div></div>

[STEPS]
?. [Day 1]
Coat plates with antibodies.
?. [Day 1]
Leave plates  at .
4 °C
?. [Day 2]
Block plates:
?. [Day 2]
Discard coating antibody and tap on paper.
?. [Day 2]
Wash with  / well 3x.
[PBS]
?. [Day 2]
Add .
[culture media]
?. [Day 2]
Incubate at  for .
37 °C
?. [Day 2]
Harvest cells from culture wells (14 day expansion) with pipette (pipetting up and down), centrifuge and count.  ABCDEFGH1#DonorStimulationCells/mLVol (mL)Total # Cells% viabilityTotal Volume to get 2x10^6/mL (uL)21
ABCDEFGH1#DonorStimulationCells/mLVol (mL)Total # Cells% viabilityTotal Volume to get 2x10^6/mL (uL)21
?. [Day 2]
Prepare stimuli dilutions:  ABCDE1StimuliStock Conc.Working Conc. (2X final conc.)V stimuli (uL)V media (uL)2Neg100%(to match max DMSO of pools)3PHA1 mg/ml20 ug/mL4Peptide pool10 ug/mL
ABCDE1StimuliStock Conc.Working Conc. (2X final conc.)V stimuli (uL)V media (uL)2Neg100%(to match max DMSO of pools)3PHA1 mg/ml20 ug/mL4Peptide pool10 ug/mL
?. [Day 2]
Plate /well each of stimuli and PBMCs.
50 µl
?. [Day 2]
Incubate plates for  at .
[,  5% CO2]
?. [Day 2]
Plate layout:  ABCDE1(1-3)(4-6)(7-9)(10-12)2APeptide pool 1Peptide pool 23BPHAPHA4CNeg for peptide pool 1Neg for peptide pool 25D6E7F8G9H
ABCDE1(1-3)(4-6)(7-9)(10-12)2APeptide pool 1Peptide pool 23BPHAPHA4CNeg for peptide pool 1Neg for peptide pool 25D6E7F8G9H
?. [Day 3]
Remove the cells by emptying the plate and wash 5 times with /well (plate washer).
[PBS]
?. [Day 3]
Dilute the detection antibodies, per full plate: NEW SYSTEM  ABCDE1Component[Starting][Final] Conc.AmountDilutions2IFNg: 7-B6-1-FS-BAM--50 ul1:2003IL-5: 5A10-WASP--50 ul1:2004IL-10: 12G81 mg/ml2 ug/ml20 ul1:5005PBS-0.1% BSA10 mL
ABCDE1Component[Starting][Final] Conc.AmountDilutions2IFNg: 7-B6-1-FS-BAM--50 ul1:2003IL-5: 5A10-WASP--50 ul1:2004IL-10: 12G81 mg/ml2 ug/ml20 ul1:5005PBS-0.1% BSA10 mL
?. [Day 3]
Add /well and incubate for  at  in the dark.
100 µl
37 Room temperature
?. [Day 3]
Wash 5 times with /well.
[PBS]
?. [Day 3]
Dilute the fluorophores, per full plate:NEW SYSTEM  ABCDE1Component[Starting][Final] Conc.AmountDilutions2IFNg: anti-BAM-490--50 ul1:2003IL-5: anti-WASP-640--50 ul1:2004IL-10: SA-550 1:200--50 ul1:2005PBS-0.1% BSA10 mL
ABCDE1Component[Starting][Final] Conc.AmountDilutions2IFNg: anti-BAM-490--50 ul1:2003IL-5: anti-WASP-640--50 ul1:2004IL-10: SA-550 1:200--50 ul1:2005PBS-0.1% BSA10 mL
?. [Day 3]
Incubate at  in the dark for .
37 Room temperature
?. [Day 3]
Wash 5 times with /well.
[PBS]
?. [Day 3]
Empty the plate and add /well and leave the plate for  at .
[Fluorescence enhancer-II]
37 Room temperature
?. [Day 3]
Empty the plate and remove residual Fluorescence enhancer by firmly tapping the plate against clean paper towels.
Do not wash in the sink.
?. [Day 3]
Remove the underdrain (the soft plastic under the plate).
?. [Day 1]
Perform all work in laminar flow hood.
?. [Day 1]
Coat plate with  per well (according to table below):  ABCDE1[Starting][Final] ConcAmountDilutions2mouse anti-human IFN-y antibody Clone 1-D1k Mabtech1 mg/ml5 ug/ml25 uL1:2003mouse anti-human IL-5 antibody TRFK5 Mabtech1 mg/ml5 ug/ml25 uL1:2004mouse anti-human IL-10 antibody1 mg/ml10 ug/ml50 uL1:1005PBS: GIBCO BRL 10010-0235 mL-
[antibody diluted in PBS]
ABCDE1[Starting][Final] ConcAmountDilutions2mouse anti-human IFN-y antibody Clone 1-D1k Mabtech1 mg/ml5 ug/ml25 uL1:2003mouse anti-human IL-5 antibody TRFK5 Mabtech1 mg/ml5 ug/ml25 uL1:2004mouse anti-human IL-10 antibody1 mg/ml10 ug/ml50 uL1:1005PBS: GIBCO BRL 10010-0235 mL-
?. [Day 1]
Discard MeOH and wash plate 3x with.
[sterile water]
?. [Day 1]
Prewet plates with / well
[70% MeOH]
?. [Day 1]
Use IPFL for Fluorospot (Mabtech).
?. [Day 3]
Leave the plate in the dark to dry; plate should be completely dry before analysis.
?. [Day 3]
Inspect and count spots in a Fluorospot reader.
?. [Day 3]
Store plate in the dark at .
0 Room temperature