Killing K562 Cells by Peripheral Blood Mononuclear Cells Exposed to Tobacco Product Preparations

Protocollo

1. K562 Killing Assay

NOTE: K562 cells should be grown in culture at 37 °C and 5% CO₂ with RPMI complete medium until they reach 80% confluence before the assay.

1. Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 µl of DMSO to the vial.
2. Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 µl/well to achieve the desired equi-nicotine units or nicotine concentrations for each well.
   CAUTION: Nicotine is acutely toxic and environmentally hazardous.
3. Add 100 µl of PBMCs into RPMI complete medium at a concentration of 1.5 x 10⁶ cells/well. The total volume of cells with WS-CM or nicotine will be 200 µl/well.
4. Cover the plate and incubate for 3 hr at 37 °C and 5% CO₂.
5. Wash the K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.
6. Resuspend the cells with 10 ml of DPBS and count the K562 cells.
7. Prepare a CFSE working solution by adding 1 µl of CFSE stock solution to 1 ml DPBS.
8. Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 - 2 x 10⁷ cells. Vortex and incubate precisely for 2 min at RT.
9. Immediately add 200 μl of FBS. Vortex and incubate precisely for 2 min at RT.
10. Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.
11. Remove the RPMI supernatant, and break the pellet, and resuspend the cells with 10 ml of RPMI complete medium, and count the CFSE-labeled K562 cells.
12. Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 µl of RPMI complete medium and repeat the washing step one more time.
13. Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 10⁶ PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 °C and 5% CO₂.
14. Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.
15. Add 200 µl of running buffer and repeat the washing step described in step 14 one more time.
16. Add 95 µl of running buffer followed by 5 µl of 7AAD to each well for a total volume of 100 µl/well. Incubate the plate in the dark at RT for 15 min.
17. Add 100 µl of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on the flow cytometer.
18. Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.

Materiali

Name	Company	Catalog Number	Comments
12 X 75 tubes	BD Falcon	352058
15 ml conical tubes	Corning	430790
2 mL Microtubes	Axygen	MCT-150-C-S
50 ml conical tubes	Corning	430828
500 ml bottle	Corning	430282
7AAD	BD Pharmingen	559925
96-well flat bottom plate	Termo Nunc	439454
96-well round bottom plates	BD Falcon	353077
Cell culture hood	Thermo Scientific	1300 Series A2
Centrifuge	Eppendorf	58110R
CFSE	Molecular Probes Life Technologies	C34554
Cluster tubes	Corning	4401	Harmful if swallowed, carcinogen
DMSO (Dimethyl sulfoxide )	Sigma-Aldrich	D8418
DPBS	Lonza	17-512F
FBS	Sigma-Aldrich	F2442
Filter unit	Nalgene	156-4020
Flow Cytometer	BD Biosciences	FACS Canto II	8 colors at Ex 405 nm and Em785 nm.
Flow Cytometer	BD Biosciences	FACS Calibur	4 colors at Ex 495 nm and Em 785 nm.
Flow cytometry analysis software	Tree Star	FlowJo
Nicotine	Sigma-Aldrich	N3876	Acute toxicity, Environmental hazard
Parafilm	Bemis	"M"
Paraformaldehyde	Sigma-Aldrich	P6148	Flammable, Skin irritation
Pen/strep	Gibco Life Technologies	15140-122
RPMI 1640	Gibco Life Technologies	11875-093
Running buffer	MACS Running Buffer, Miltenyi Biotech	130-091-221
Transfer pipette	Fisher Scientific	13-711-20
Tris Base	Sigma-Aldrich	T1503