In vitro quantitation of Theileria parva sporozoites for use - TropMed ...

152 Appendices
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B. REFINED PROTOCOL
Day -1
Peripheral Blood Mononuclear Cells isolation
The same as in Appendix I.A
PBMC counting:
The same as in Appendix I.A
After counting,
Add medium to obtain 6*10 6 PBMC/ml
Transfer 12 ml PBMC in 50 ml culture bottles (1 bottle will be for 1 plate)
Keep the PBMC overnight in a refrigerator, the bottles lying on their large surface.
Day 0
In vitro titration
(An example comparing glycerol and sucrose stabilates is given here)
Fill columns 2-12 top half of the plate, with 50 µl of stabilate diluent (7.5% glycerol)
Fill columns 2-12 bottom half of the plate, with 50 µl of stabilate diluent (0.3M sucrose)
Fill first column with 150 µl of respective stabilate (dil 1) (4 replicates per stabilate)
Make 1.5x serial dilutions of stabilate by transferring 100 µl from column to column (using a
multichannel pipette).
Pipette-mix about 7 times at each dilution step including dil 1.
Discard 100µl from the last column
Add 50 µl PBL in each well using a multichannel pipette.
Pipette-mix about 3 times (Use a fresh set of tips for each column)
Incubate the plate for 30 min at 37°C in CO 2 incubator (record time)
Shake plate for 1 min using a Titertek ® shaker (Flow Laboratories) at 10 Hz and return to
incubator for another 20 min
Centrifuge the microplate at 210 g for 10 min
Decant supernatant