Jun 07, 2020
UT Southwestern - Staining Melanoma Cells for Flow Cytometry
This protocol is a draft, published without a DOI.
- Arin Aurora1,
- Sean Morrison1
- 1UT Southwestern, Morrison Lab
- NCI PDMC consortium
Protocol Citation: Arin Aurora, Sean Morrison 2020. UT Southwestern - Staining Melanoma Cells for Flow Cytometry. protocols.io https://protocols.io/view/ut-southwestern-staining-melanoma-cells-for-flow-c-bg9gjz3w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2020
Last Modified: June 07, 2020
Protocol Integer ID: 37896
STAINING CELLS FOR FLOW CYTOMETRY
STAINING CELLS FOR FLOW CYTOMETRY
Adjust staining volume and antibody concentration to the number of cells. Concentrations listed are for 5x106 cells in a 50ml reaction. Do not exceed 106 cells/10ml.
- mLin APC: Ter119 (1/100), CD31(1/100), CD45(1/200)
- HLA FITC: HLA-ABC (1/5)
Add antibodies to tube and incubate covered on ice for 00:20:00
Wash 1x with 1 mL staining medium and spin down 220 rpm, 4°C, 00:04:00 . Aspirate supernatant
DAPI staining: resuspend samples in DAPI 1:2000 to gate out dead cells. Stock in made in water (1mg/ml)
FACS
FACS
Adjust final sample volume to run at 6000 events/sec at maximum and then run. If clogging becomes an issue, then filter through cut pieces of filter
| Xenograft stain (50ml volume) | 1x | 5x | |
| HLA- PE or FITC (1/5) | 10ml | 50ml | |
| mCD45 APC (1/200) | 0.3ml | 1.5ml | |
| mCD31 APC (1/100) | 0.5ml | 2.5ml | |
| mTer119 APC (1/100) | 0.5ml | 2.5ml |
| Human stain (50ml volume) | 1x | 5x | |
| HLA- FITC (1/5) | 10ml | 50ml | |
| ISO PE or P75 PE (1/5) | 10ml | 50ml | |
| Glyc-A APC (1/2000) | 0.025ml | 0.125ml | |
| hu-CD31 APC (1/800) | 0.063ml | 0.315ml | |
| hu-CD45 APC (1/5) | 10ml | 50ml |