TITLE: Eosin-5’-maleimide (EMA) binding test with fluorecent beads
AUTHORS: Andreas Glenthoej, Jesper Petersen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The flow cytometry-based eosin-5’-maleimide (EMA) binding test is used for reliable diagnostics of hereditary spherocytosis, a relatively common hereditary anemia. </div><div class = "text-block">In this modified version of the EMA-binding test, we utilize commercially available rainbow beads to overcome the the need for three to six healthy controls. However, two healthy controls are still used in this protocol as an extra safety measure.</div></div>

[STEPS]
?. [Run samples on the Flow Cytometer]
Prepare FACS-tubes for beads:Label 3 tubes for beads and add 500 µl PBS to each.Add 1 drop of beads to each tube (Calibration beads, Blank beads, and rainbow beads).Calibration beads and blank beads are part of a calibration kit from Agilent (#K011011-2)Rainbow beads are puchased separately (BD Biosciences, #556298)
?. [Prepare samples]
Remove a tube of stock EMA () aliquot from the freezer to thaw in a dark place @  .
0 Room temperature
?. [Prepare samples]
Prepare samples
?. [Preparation of EMA Reagent batches]
Preparation of stock of EMA-reagent:
?. [Preparation of EMA Reagent batches]
Dissolve  EMA in  PBS.
10 mg
20 mL
?. [Preparation of EMA Reagent batches]
Transfer aliquots of EMA solution to each tube.
250 µl
?. [Preparation of EMA Reagent batches]
Store in freezer.Aliquots of frozen dissolved EMA dye should be stable for at least six months. Aliquots should be discarded after use (i.e. not frozen agin for later use).
?. [Prepare samples]
Label a 14 ml tube for each sample  0.9% NaCl is added to each tube  of mixed blood is added to its corresponding tubeMix/Vortex tubes  Discard supernatant 0.9% NaCl is added to each tube of mixed blood is added to its corresponding tubeMix/Vortex tubes  Discard supernatant
10 mL
100 µl
Centrifuge: 1500 34, 5 min, 22 32
?. [Prepare samples]
Wash twice:Add  0.9% NaCl Mix/Vortex  Discard supernatant
10 mL
Centrifuge: 1500 34, 5 min, 22 32
?. [Prepare samples]
Transfer  of washed packed red cells to a new (labelled) 1.5 mL eppendorf tube
5 µl
?. [Prepare samples]
Add  of EMA stock solution Incubate for  @ Mix every
25 µl
[in a dark place]
?. [Prepare samples]
Centrifuge: 1500 34, 30 s
?. [Prepare samples]
Add  PBS
500 µl
?. [Prepare samples]
Transfer  of labelled red cells to the new tube
25 µl
?. [Prepare samples]
Wash the labelled RBC's 3 times:Add PBS with 0.5% BSAMix/Vortex  Discard supernatant
3 mL
Centrifuge: 1500 34, 5 min, 22 32
?. [Prepare samples]
Samples are now ready to be run on the flow cytometer.
?. [Run samples on the Flow Cytometer]
For each patient sample:Start running the Calibration beads and adjust the voltage of the PMT in such a way that the fluorescence FL1 is approximately 50,000 (or as close hereto as possible). Acquire 15,000 events.Run Blank beads and acquire 15,000 events.Run Rainbow beads and acquire 15,000 events.Run Control samples and acquire 15,000 events.Run Patient sample and acquire 15,000 events.
?. [Run samples on the Flow Cytometer]
Setup flow cytometer (Canto II):Set it to LOG-mode for FSC, SSC, and FL1 Apply a threshold of 5000 for the FSC.
?. [Analyze results]
The analysis of the results can be done differently.
?. [Prepare samples]
Add PBS with 0.5% BSA
500 µl