6/8/2018

In Vitro Immunogenic Cell Death (ICD) Stimulation with 4T1.2 cells

Purpose:
To stimulate immunogenic cell death in cancer cell lines using doxorubicin or IFNa/retinoic acid and verify ICD by FACs

Materials:
Mouse IFNa (Miltenyi Cat# 130-093-131)
9-cis-Retinoic acid (Sigma Cat# R4643)
Doxorubicin hydrochloride (Sigma D1515)
Anti-CD47 FITC miap301 Biolegend 0.5mg/mL (1:100 dilution)
Anti-Calreticulin AF647 ab196159 Abcam 0.5mg/mL (1:50)
Isotype Rabbit IgG AF647 ab199093 Abcam 0.5mg/mL (1:50)
Annexin V PacBlue 640918 Biolegend 80ug/mL (5uL/test)
Annexin V Binding buffer (defined salt conditions and Ca2+, which is required for AnnV binding)
7-AAD PCP5.5 Cat#420404 50ug/mL (5uL/test)

Methods:
Day 0 Plate cells in T25 flask with following treatments
doxo RA IFNa
1x doxo 100nM
1x RA/IFN 1uM 1000IU/mL
No Treatment
Incubate at 37C 5% CO2 for 4 days

Day 4 Stain Cells for FACS

1 Collect floating cells in supernatant of flasks into 50mL tube
2 Wash cells with 3mL PBS and collect into same tube
 3 Dissociate adherent cells using TrypLE (1mL/T25 flask)
Note: Do not use trypsin/EDTA as the surface calreticulin will be digested
4 Inactivate TrypLE with 4mL complete media and collect cells into 50mL tube
Be careful when harvesting to prevent over-trypsinisation and avoid physical stress/bubbles
Excessive stress can induce non-treatment specific apoptosis or loss of surface antigens
5 Centrifuge 1200 rpm for 5 min at 4C
6 Wash again with PBS
7 Incubate cells in cold blocking buffer (2% FBS in PBS) for 15 min at 4C in the dark
8 Aliquot 50uL of ~0.5e6 cells (or 1/5 of T25) from each treatment to FACs tubes for staining
9 Add 1/5 cells to FACs tubes and stain
Samples (full stain) Single color Unstained controls
No treatment anti-CRT AF647 NT cells
doxo 7-AAD PCP5.5 doxo cells
doxo + Isotype Annexin V Pac Blue
RA/IFNa
RA/IFNa + Isotype Fluorescence Minus One
7AAD + AnnV (No CRT)

Note: When staining with CRT-AF647, use AnnV-FITC and DAPI instead of 7AAD
Note: When staining with HSP70-AF488, use AnnV-PacBlue and 7AAD
10 Stain with CRT (1:100) antibodies by making mastermix and adding 50uL mastermix to cells (total 100uL)
Incubate 30 min at 4C in the dark
11 Wash cells with PBS
12 Wash cells with 0.5mL Annexin V binding buffer
13 Pour out supernatant (leaving ~100uL of cells)
14 Stain Annexin V (4uL) and 7-AAD (4uL) viability dyes (or 2uL DAPI) to sample tubes and single colors
Incubate 15 min covered at room temp
Do not wash viability stains out
15 Add 100uL Annexin V Binding Buffer to sample tubes and acquire on FACs machine
Previous Settings:
FSC: 200 (gain 1) FL1 FITC CD47: 400
SSC: 500 (gain 10) FL2 PE:
FL4 7AAD: 580
FL6 CRT AF647: 500
FL9 AnnV PacBlue: 280
(Use neutral density filter and no threshold. Run at least 100,000 evts per tube on medium).

Nanoparticle Settings (for Kelvin)

Previous Settings:
488nm Blue 638nm Red 405nm Violet
FSC: 100 (gain 1) FL1 FITC AnnV: 330 FL6 AF647: 520 FL9 PacBlue/DAPI: 280
SSC: 400 (gain 10) FL2 PE: 380 FL7: 540 FL10: 250
FL3: 400 FL8: 530
Discriminator FS 20 FL4 7AAD: 520
Small FL5: 580
Neutral density filter
Run at least 100,000 evts per tube on medium