Aug 19, 2020
HTAPP_Depletion of CD45+ cells from single-cell suspension for single-cell RNA-seq
- Isaac Wakiro1,2,
- Sara Napolitano1,2,
- Sébastien Vigneau1,
- Asaf Rotem1,2,
- Orit Rozenblatt-Rosen3,2,
- Bruce Johnson1,2,
- Aviv Regev3,4,5,6
- 1Dana-Farber Cancer Institute;
- 2Human Tumor Atlas Pilot Project;
- 3Broad Institute;
- 4Human Tumor Atlas Project;
- 5Massachusetts Institute of Technology;
- 6Howard Hughes Medical Institute
- NCIHTAN
Protocol Citation: Isaac Wakiro, Sara Napolitano, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev 2020. HTAPP_Depletion of CD45+ cells from single-cell suspension for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bjxjkpkn
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: August 19, 2020
Last Modified: August 19, 2020
Protocol Integer ID: 40651
Abstract
In some tumor specimens the proportion of malignant cells is relatively low and the that of immune cells high. In these cases, we considered strategies to deplete CD45+ immune cells as a way to both enrich for epithelial cells without using specific epithelial cells markers, and to maintain the stromal cells.
The protocol described here is used for the depletion of CD45+ expressing immune cells from human tumor-derived single-cell suspensions. It is adapted from the CD45 MicroBeads MACS Separation protocol from Miltenyi Biotec.
For the Human Tumor Atlas Pilot Project (HTAPP), CD45 depletion was used to increase the recovery of malignant epithelial cells from lung non-small cell adenocarcinoma samples in which the proportion of these types of cells was low.
Description of this protocol and guidance for testing and selecting methods for processing different tumor and sample types can be found in Slyper et al.
Guidelines
- Keep sample on ice and use cold reagents unless noted otherwise.
- Report information as indicated in the protocol.
Materials
MATERIALS
UltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616
PBS pH 7.4Thermo Fisher ScientificCatalog #10010049
BSACell Signaling TechnologyCatalog #9998S
Trypan Blue Solution 0.4% Sterile-filtered Sigma AldrichCatalog #T8154
Flex-Tube® 1.5 mL PCR clean colorlessEppendorfCatalog #022364120
Tips RT-LTS-A-1000µL-/F-768/8RaininCatalog #30389212
Centrifuge 5430 R refrigerated with Rotor FA-45-30-11 incl. rotor lid keypad 120 V/50 – 60 Hz (US)EppendorfCatalog #022620601
Tips RT-LTS-A-10µL-/F/L-960/10RaininCatalog #30389226
Tips RT-LTS-A-200µL-/F/L-960/10RaininCatalog #30389240
Falcon® 15 mL High Clarity PP Centrifuge Tube Conical Bottom with Dome Seal Screw Cap Sterile 50/Rack 500/CaseCorningCatalog #352097
NanoEnTek Inc. Disposable HemocytometerWestnetCatalog #C-CHIP
MiniMACS SeparatorMiltenyi BiotecCatalog #130-042-102
MACS MultiStandMiltenyi BiotecCatalog #130-042-303
MS ColumnsMiltenyi BiotecCatalog #130-042-201
CD45 MicroBeads humanMiltenyi BiotecCatalog #130-045-801
Trypan Blue solutionMillipore SigmaCatalog #T8154
Stericup Quick Release-GP Sterile Vacuum Filtration SystemMillipore SigmaCatalog #S2GPU02RE
Sorvall Legend RT Refrigerated Tabletop CentrifugeThermo Scientific
Safety warnings
Follow general lab safety and institutional guidelines when working with sharps and human derived samples.
Before start
- Set centrifuges to 4˚C.
- Label four 15 mL conical tubes as "Supernatant", "Wash", "CD45-" and "CD45- Supernatant", and two 1.5 mL Eppendorf tubes as "CD45-". Optionally, if you plan to also collect the CD45+ fraction for further analysis, label two 15 mL conical tubes as "CD45+" and "CD45+ Supernatant", and one 1.5 mL Eppendorf tube as "CD45+". Keep tubes on ice. Supernatants are collected to enable, if needed, the recovery by centrifugation of cells that have failed to pellet or were accidentally aspirated (e.g., if the final yield is too low).
- Prepare MACS buffer: PBS with 0.5% BSA and 2 mM EDTA (using Cell Signaling Technology BSA #9998S). Sterile filter, degas, and keep on ice. This solution can be prepared in advance and stored at 4˚C for several weeks.
- Prepare PBS with 0.4% BSA (using Thermo Fisher Scientific UltraPure BSA #AM2616) and keep on ice. This solution can be prepared in advance and stored at 4˚C for several weeks.
Initial Quality Control
Initial Quality Control
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
Count and record the number of viable single-cells, dead single-cells, cell doublets, and whether debris are present, then calculate additional quality control metrics below. Take picture if possible.
| Initial Cell Suspension | ||
| Number of Viable Single Cells Counted | ||
| Number of Dead Single Cells Counted | ||
| Number of Cell Clumps or Doublets Counted | ||
| Concentration of Viable Single Cells (cells/µL) | ||
| Concentration of Dead Single Cells (cells/µL) | ||
| Concentration of Cell Clumps or Doublets (doublets/µL) | ||
| Volume of Single Cell Suspension (µL) | ||
| Total Number of Viable Single Cells | ||
| Proportion of Single Cells that are Viable (%) | ||
| Proportion of Cell Clumps or Doublets (%) | ||
| Description of debris (if any) |
Note
Insert Picture:
Magnetic Labeling
Magnetic Labeling
Centrifuge cell suspension in a 1.5 mL Eppendorf tube at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
500 x g, 4°C, 00:04:00
Carefully transfer supernatant to the 15 mL “Supernatant” tube kept on ice without disturbing the pellet.
On ice
If the cell pellet contains less than 107 cells, resuspend in 80 µL MACS buffer. If the cell pellet contains more than 107 cells, resuspend in 80 µL MACS buffer per 107 cells.
On ice
Note
Volume of MACS Buffer Used (µL):
Add 20 µL CD45 microbeads per 80 µL MACS buffer.
On ice
Note
Volume of Microbeads Used (µL):
Incubate in 4°C refrigerator for 15 minutes.
00:15:00 Labeling
4 °C Refrigerator
During the incubation, insert an MS column into the MiniMACS separator set on the MACS MultiStand. Prime the column with 500 µL MACS buffer, collecting the effluent in the 15 mL "Wash" tube.
Room temperature
Following incubation (Step 7), add 1 mL cold MACS buffer per 100 µL suspension.
On ice
Note
Volume of MACS Buffer Used (µL):
Centrifuge suspension at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
500 x g, 4°C, 00:04:00
Carefully transfer supernatant to the 15 mL “Supernatant” tube kept on ice without disturbing the pellet.
On ice
If the cell pellet contains less than 108 cells, resuspend in 500 µL MACS buffer. If the cell pellet contains more than 108 cells, resuspend in 500 µL MACS buffer per 108 cells.
On ice
Note
Volume of MACS Buffer Used (µL):
Collection of the CD45- fraction
Collection of the CD45- fraction
Position the 15 mL "CD45-" tube under the MS column, discarding the "Wash" tube.
Room temperature
Transfer cell suspension (up to 2x108 cells) to the primed MS column on the MiniMACS separator and collect the flow-through in the 15 mL "CD45-" tube.
Room temperature
Wash the MS column by adding 500 µL MACS buffer and collecting the flow-through in the 15 mL "CD45-" tube. Repeat wash two more times.
Room temperature
Transfer the "CD45-" tube on ice.
On ice
[Optional] Collection of the CD45+ fraction
[Optional] Collection of the CD45+ fraction
Remove the MS column from the MiniMACS separator and position it on top of the 15 mL "CD45+" tube.
Room temperature
Add 1 mL of MACS Buffer to the column and immediately elute the CD45+ cells by firmly pushing the plunger into the column.
Room temperature
Transfer the "CD45+" tube on ice.
On ice
Cell Concentration
Cell Concentration
Transfer the CD45- fraction to two 1.5 mL "CD45-" Eppendorf tubes on ice. (If a CD45+ fraction has been collected, transfer it to one 1.5 mL "CD45+" Eppendorf tube on ice.)
On ice
Centrifuge cell suspensions at 500 g for 4 minutes in 4˚C pre-cooled centrifuge.
Tip: If the number of cells is expected to be small (e.g., less than 50,000 cells), higher recovery may be obtained by centrifugation for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to but not exceeding 11,000 g (do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death).
500 x g, 4°C, 00:04:00
Carefully transfer supernatant from the "CD45-" tubes to the 15 mL “CD45- Supernatant” tube kept on ice without disturbing the pellet. (If a CD45+ fraction has been collected, transfer the supernatant from the "CD45+" tube to the 15 mL "CD45+ Supernatant" tube kept on ice, without disturbing the pellet.)
On ice
Resuspend and combine both CD45- cell pellets in a total volume of 50 µL cold PBS with 0.4% BSA. (If a CD45+ fraction has been collected, resuspend the CD45+ pellet in 50 µL cold PBS with 0.4% BSA.)
On ice
Final Quality Control
Final Quality Control
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
Count and report the number of viable single cells, dead single cells, cell doublets or clumps, and whether debris are present, then calculate additional quality control metrics below. Take picture if possible.
| CD45- Fraction | CD45+ Fraction (if collected) | ||
| Number of Viable Single Cells Counted | |||
| Number of Dead Single Cells Counted | |||
| Number of Cell Clumps or Doublets Counted | |||
| Concentration of Viable Single Cells (cells/µL) | |||
| Concentration of Dead Single Cells (cells/µL) | |||
| Concentration of Cell Clumps or Doublets (doublets/µL) | |||
| Volume of Single Cell Suspension (µL) | |||
| Total Number of Viable Single Cells | |||
| Proportion of Single Cells that are Viable (%) | |||
| Proportion of Cell Clumps or Doublets (%) | |||
| Description of debris (if any) |
Note
Insert Picture for CD45- fraction:
Insert Picture for CD45+ fraction (if collected):
Loading on 10x
Loading on 10x
If necessary, adjust the concentration of the CD45- fraction before proceeding to loading on 10x Genomics Single-Cell RNA-seq system, following 10x Genomics recommendations.
Tip: 8,000-10,000 live cells are typically loaded per channel. Optimal cell recovery is achieved for concentrations between 800 and 1,200 cells/µL but deviations from that range are acceptable (see 10x Technical Note on this topic). Furthermore, it is recommended that viability be higher than 60% and the proportion of cell clumps lower than 5%.
On ice
Report the information listed below about loading on 10x Genomics Single-Cell RNA-seq system, including the number and concentration of cells per channel.
Note
Time of Loading:
Person Loading:
Single-Cell RNA-seq Kit Used:
Concentration of Viable Cells Loaded (cells/µL):
Number of Cells Loaded per Channel:
Number of Channels Loaded: