Nov 02, 2022
Version 2
Cell preparation for scRNA-Seq from diluted bodily fluids V.2
- Linas Mazutis1,
- Vaidotas Kiseliovas1,
- Adrienne Boire2
- 1Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;
- 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
External link: https://doi.org/10.1126/science.aaz2193
Protocol Citation: Linas Mazutis, Vaidotas Kiseliovas, Adrienne Boire 2022. Cell preparation for scRNA-Seq from diluted bodily fluids. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg366r1g25/v2Version created by Linas Mazutis
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: November 02, 2022
Last Modified: November 02, 2022
Protocol Integer ID: 72215
Abstract
Efficient isolation of cells from complex bodily fluids is a crucial step for many biological and biomedical applications. Yet, it can become a particularly challenging when only a small fraction of cells is dispersed in a large volume of fluid, e.g. ~ 1000-10000 cells / 10 mL. In such cases a conventional wisdom would suggest the use of canonical 15 or 50 ml tubes and centrifugation forces at 500-800g to pellet the cells. However, hard centrifugation can damage the cells and rupture their membrane, while at lower centrifugation speeds the sedimentation rates are slow and might be insufficient to form a cell pellet. Moreover, intrinsic variability of cells in terms of size (6-20 µm) and density (1.06-1.29g/mL) may reduce the pelleting and recovery yields.
When using swinging bucket rotors and large sample volumes (> 5 mL) the sample sedimentation run times can become very long due to the increased sedimentation path length (distance that cells need to travel to reach bottom of the tube). On another hand, in fixed angle rotors the cells will travel only a short distance before hitting the wall of the tube and as a result will migrate down the wall forming a long trail until reaching the bottom of the tube, which will preclude formation of a tight pellet. Overall, when working with fragile cell samples preference should be given to lower centrifugal forces and shorter centrifugation times. These two requirements can be fulfilled by using smaller tubes as the shorter sedimentation path length will shorten the distance that cells need to travel to reach bottom of the tube.
Given these considerations this protocol describes the isolation of viable primary cells from diluted suspensions of bodily fluids. The protocol exemplifies the isolation of cells (immune and cancer cells) from cerebrospinal fluid where the concentration of cells is in the order of 0-20 cells / µl.
Materials
HemocytometerThermo Fisher ScientificCatalog #A25750
Microcentrifuge tubes (1.5 ml Protein LoBind tubes)EppendorfCatalog #022431081
Phosphate Buffered Saline Gibco, ThermoFisherCatalog #10010023
Trypan Blue Solution 0.4%Thermo Fisher ScientificCatalog #15250061
10% (w/v) BSA filtered through 0.2 μm membraneSigmaCatalog #A7906-100G
Nuclease-free WaterThermo Fisher ScientificCatalog #AM9937
Falcon, conical centrifuge tubes CorningCatalog #352070
Chromium Single Cell 3 GEM Library & Gel Bead Kit v310x GenomicsCatalog #1000075
Chromium Chip B Single Cell KitCatalog #1000073
Chromium i7 Multiplex KitCatalog #120262
Equipment
Countess II
NAME
Cell counter
TYPE
Thermo Fisher Scientific
BRAND
AMQAX1000
SKU
Equipment
Swinging bucket cooling centrifuge
NAME
Centrifuge
TYPE
Fisher Scientific
BRAND
SorvallTM LegendTM X1 Centrifuge Series
SKU
Equipment
Ice bucket
NAME
.
BRAND
.
SKU
Sample retrieval
Sample retrieval
1h
1h
Typically one could expect to have 3-5 ml of starting fluid.
1h
Concentrating cells
Concentrating cells
22m
22m
- Divide the fluid from step #1 to microcentrifuge tubes at 0.5 ml per tube.
Note: When working with large volume >5 ml it may be inconvenient to handle multiple tubes. A fluid volume per single tube can be increased to 0.75-1.0 ml.
- Transfer the tubes to a swinging bucket centrifuge and spin at 300 g for 5 min at 4 ºC with breaks off. Once centrifugation is complete the cell pellet will form at the bottom of the tube, but it may be invisible to the eye.
- Place tubes on ice.
7m
- Carefully aspirate the supernatant from each tube leaving ~50 µl on top of cell pellet.
- Gently disperse the cell pellet in each tube by slowly pipetting 5-times using P200 pipette.
- Combine the cell suspensions into one microcentrifuge tube.
- Add ice-cold 1X PBS buffer supplemented with 1% BSA to the final volume of 1 ml.
5m
- Divide 1 ml suspension into two tubes, each equal volume (0.5 ml).
- Centrifuge the tubes at 300 g for 5 min at 4 ºC in a swinging bucket centrifuge with breaks off.
- Place microcentrifuge tubes on ice.
7m
- Carefully aspirate the supernatant in each tube leaving 20-30 µl on top of pellet.
- Disperse the cell pellet in each tube by slowly pipetting 10-times using P20 pipette.
- Combine the cell suspensions into one microcentrifuge tube.
- At this step cell suspension should be ~40-60 µl.
3m
Counting cells
Counting cells
5m
5m
- Count the cells on a hematocytometer by mixing 5 µl of cell suspension with 5 µl of 0.4% Trypan Blue dye:
- If the cell count is higher than 600 cells / 1 µl proceed to scRNA-Seq step.
- If the cell count is lower than 600 cells / 1 µl then centrifuge cells again at 300g for 5 min at 4 ºC in a swinging bucket centrifuge with breaks off and discard the requirement supernatant volume to concentrate the cells. For example, if the starting volume is 100 µl one may choose to discard 70 µl and resuspend cell pellet in the remaining 30 µl of supernatant buffer (e.g. 1X PBS + 1% BSA).
5m
Barcoding cells for scRNA-Seq
Barcoding cells for scRNA-Seq
20m
20m
20m