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

More documents

Recommendations

Info

Appendices 151 _______________________________________________________________________________________________ Prepare 4 x 12 = 48 eppendorf tubes (4 rows x 12 columns) Fill columns 2-12 of two rows with 150 µl of stabilate diluent -7.5% glycerol Fill columns 2-12 of next two rows with 150 µl of stabilate diluent – 0.3M sucrose Fill first column with 450 µl of respective stabilate (dil 1) Make 1.5x serial dilutions of stabilate by transferring 300 µl from column to column Add 150 µl PBMC in each tube Let incubate the tubes for 1 h in thermoshakers (37°C, 1000 rpm) (record time) Centrifuge the tubes at 210 g for 10 min Decant supernatant Resuspend the cells in 0.6 ml (per tube) culture medium Transfer 0.2 ml from each tube in 2 wells of a 96-well plate Label the plates Transfer to CO 2 incubator (37°C ) - (record time) Leave undisturbed for 10 days Day 10 Destroy fungi contaminated cells with 10N NaOH. Check cell cultures for clumps Take cytospins: mount slides with a Filter Card “Shandon”, centrifuge 50 to 100 µl at 215 g for 5 minutes. Stain with May-Grunwald / Giemsa’s stain Score cultures for the presence or the absence of schizonts
152 Appendices ______________________________________________________________________________________________ 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
Page 1 and 2:
Victor Mbao In vitro quantitation o
Page 3 and 4:
II Front cover photograph Copyright
Page 6 and 7:
V ACKNOWLEDGEMENTS This work was ma
Page 8 and 9:
VII TABLE OF CONTENTS ACKNOWLEDGEME
Page 10:
IX APPENDIX I .....................
Page 14 and 15:
General Introduction 13 ___________
Page 16 and 17:
General Introduction 15 ___________
Page 18:
Chapter 1 - Quantitation of Theiler
Page 21 and 22:
20 Chapter 1: Quantitation of Theil
Page 23 and 24:
Page 25 and 26:
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35 and 36:
Page 37 and 38:
Page 39 and 40:
Page 41 and 42:
Page 44 and 45:
Chapter 2: Objectives 43 __________
Page 46:
Chapter 3 -Comparison of two Theile
Page 49 and 50:
48 Chapter 3: Theileria parva extra
Page 51 and 52:
Page 53 and 54:
Page 55 and 56:
Page 58 and 59:
Chapter 4: Suspension media and mul
Page 60 and 61:
Page 62 and 63:
Page 64 and 65:
Page 66 and 67:
Page 68:
Chapter 5 - Snap freezing of Theile
Page 71 and 72:
70 Chapter 5: Snap freezing of Thei
Page 73 and 74:
Page 75 and 76:
Page 78 and 79:
Chapter 6: Storage of Theileria par
Page 80 and 81:
Page 82 and 83:
Page 84 and 85:
Page 86 and 87:
Page 88 and 89:
Page 90:
Page 94 and 95:
Chapter 7: Lyophilisation of Theile
Page 96 and 97:
Page 98 and 99:
Page 100:
Page 104 and 105: Chapter 8: SNA and refinement of ti
Page 116: Chapter 8: SNA and refinement of ti
Page 120 and 121: Chapter 9: General Discussion 119 _
Page 126: Chapter 9: General Discussion 125 _
Page 130 and 131: References 129 ____________________
Page 148: APPENDICES
Page 151: 150 Appendices ____________________
Page 155 and 156: 154 Appendices ____________________
Page 166: SUMMARY
Page 169 and 170: 168 Summary _______________________
Page 171 and 172: 170 Summary _______________________
Page 174 and 175: Samenvatting 173 __________________
Page 176 and 177: Samenvatting 175 __________________
Page 178: Curriculum Vitae
Page 181 and 182: 180 Carriculum Vitae ______________
Page 184 and 185: List of Abbreviations 183 _________
show all

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

Create successful ePaper yourself

Delete template?

Save as template?