T-SPOT PROTOCOL The following procedures are to be followed for testing whole blood samples with the oxford immunotec’s t-spot assay. Specimen collection and handling Blood samples are collected into lithium heparin blood collection tubes with PBMCs being subsequently separated using standard method separation techniques such as ficollpaque. Note EDTA tubes are not recommended. the tubes contents must be inverted 8-10 times to ensure that the whole blood is mixed thoroughly with the anticoagulant. Store collected blood at room temperature. Blood sample should be proceed within 8hours. A patient’s cell can be pooled, if necessary to obtain sufficient cells from multiple tubes of blood which were collected and processed concurrently Day one Take out FBS,RPMI, and AIM-V and leave @ RT for 30 mins before starting the T-Spot 1. 2. 3. 4. 5. 6. Label 3 (15ml or 20ml ) tubes: A,B, and C transfer blood sample to a if blood sample is not processed after collection, invert tubes few times before transfer it. Add RPMI to A, amount of RPMI=amount of blood sample. Mix well Add 4mls of ficoll to B. Transfer slowly all blood samples from A to B without disruption of Ficoll layer by transferring on the side of the tube. Centrifuge B @ 1000 RCF(g) for 22mins at room temperature with brake 1 and acceleration 1. this is good time to prepare stripes/plate (use 4 wells for each sample). After stripes, panel A, panel B, and positive control have reached room temperature; add (on side of the well- don’t break the membrane) 50 of: AIM-V into A (and E) panel A into B (and F) Panel B into C (and G) positive control into D (and H) Tap the plate ,put in incubator until the sample is ready. 7. After the spin, pipette off the PBMCs into a fresh 15mlml tube C. The PBMCs will be the thin cloudy layer between the plasma and ficoll layer. The volume (in ml) of AIM-V=0.5-Y EXAMPLE: i. # Cells counted on hemacytomer =131. ii. 131 *5*10 = 6.55 million cells per ml of cell suspension to use. iii. (0.5*2.5)/6.55=0.19ml of cell suspension to use. iv. Remove 0.19ml of cell suspension and add to this 0.31ml of AIM-V to bring the final volume to 0.5ml 8. Pipette amount of cell suspension and AIM-V from step 19 to 0.6ml tube. 9. Slowly transfer 100µI of diluted cell to each well (change the tip). Using these calculations, when placing 100µI of cell dilution into each well, you will be placing 250,000cells into each well. 10. Incubate the plate @ 37°c with 5% CO2 for 16-20 hours. Place a wet paper under the plate. Spin the last tube (C, cell suspension) @ 350 RCF for 5mins. 11. Pour off the supernantant and add 1ml of FBS (prepared by adding 5mls of DMSO to 45mls of FBS. Replace this bottle after 1 month). Then,transfer the cell solution into new crewed cap tube and store @-20°c in 70% ethanol. Next day store cell @ -80°c Day two 1. Take substrate solution out of storage and allow it to reach room temperature. 2. Prepare a 1:200 dilution of working conjugate reagent. 50µI of working. conjugate reagent will be needed in each well. For eight wells, use 2.5µI of conjugate reagent to 500µI of PBS (1x). 3. Take the plate out and tap the plate to remove the cells 4. Dump off cell culture medium into the sink. 5. wash the plate 4 times with 1x PBS by holding the squeeze bottle upside down; spray approximately 200µI PBS into each well. Then dump off the PBS into the sink. 6. Tap the plate upside down on apaper towel to remove excess PBS. 7. Add 50 of 1:200 working conjugate reagent dilution into each well. 8. Incubate at2-8c for 60mins and then put in refrigerator, protect from light. 9. Wash the plate 4 times with 1x PBS as step 5 10. Tap the plate up side down on a paper towel to remove excess PBS. 11. Add 50 of substrate into each well. 12. Place at room temperature for 7mins. Do not walk away from the plate-watch carefully. 13.Tap plate upside down on a paper to remove substrate. 14. Wash thoroughly the plate with distilled water 4 times inside the well and on the bottoms of the wells. 15.Tap the plate upside down on a paper towel to dry.