Hs 5 cells

SUDHL-10 cells (DLBCL line) and HS-5 cells (fibroblast line) were
originally purchased from American Type Culture Collection (ATCC, Manassas, VA).
The SUDHL-10 cells were maintained in RPMI 1640 media supplemented with 10%
fetal bovine serum and 1% antibiotic-antimycotic solution (Corning Cellgro,
Manassas, VA) and the HS-5 cells were maintained in DMEM medium with 4.5 g
L⁻¹ glucose and L-glutamine (Corning Cellgro, Manassas,
VA) with serum and antibiotic-antimycotic solution.
Peripheral Blood mononuclear cells (PBMCs) were obtained from Astarte
bio (Astarte Biologics, Bothell, WA) and subsequently treated to generate
activated PBMCs. Briefly, anti-CD3 antibody was used to coat well plates at a
concentration of 10 μg mL⁻¹ for a period of 2 hours.
The cells were then seeded at a concentration of 15, 000 cells per well along
with an activation cocktail consisting of IL-2 (10 ng mL⁻¹),
IL-4 (20 ng mL⁻¹) and soluble anti-human CD-28 antibody (2
μg mL⁻¹) for two days. The plate was centrifuged and
re-treated for another two days with IL-2 (10 ng mL⁻¹) and
IL-4 (20 ng mL⁻¹). The cells were then harvested, washed and
resuspended in fresh IL-2 containing media for experiments.
All cells were grown at 37°C under 5% CO₂ in a
humidified atmosphere. Cells were routinely passaged every three days and
harvested at a density of 1 × 10⁶ viable cells
mL⁻¹.

Human bone marrow stromal HS-5 cells were purchased from American Type Culture Collection (ATCC) and cultured as above. HS-5 conditioned media was prepared by culturing HS-5 cells to 70% confluence, after which media was removed and replaced with fresh media. After 24 hr of incubation HS-5-conditionned media was collected and debris removed by centrifugation. Lymphoma cells were incubated in HS-5-conditioned media for 24 hr before treatment. For co-culture studies, lymphoma cells were incubated with HS-5 cells for 24 hours, then treated for 24 hr, after which non-adherent cells were collected and subjected to Annexin V/PI assay.