Jul 27, 2022
Version 2
PBMCs isolation from CPT™ tube V.2
- Woong-Yang Park1,
- Jay Shin2,
- Shyam Prabhakar3
- 1SMC;
- 2RIKEN;
- 3GIS
Protocol Citation: Woong-Yang Park, Jay Shin, Shyam Prabhakar 2022. PBMCs isolation from CPT™ tube. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxeqr4v8j/v2Version created by Nastia M
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: May 04, 2022
Last Modified: July 27, 2022
Protocol Integer ID: 61985
Keywords: PMBC isolation, blood collection, centrifugation,
Abstract
This protocol details the procedure for collection and isolation of blood samples using CPT tubes.
Attachments
Guidelines
Materials
Materials:
Chemicals and Reagents:
Fetal Bovine SerumSigma AldrichCatalog #F2442
PBS, pH 7.4Thermo FisherCatalog #10010049
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
CryoStor® CS10Stemcell TechnologiesCatalog #07955
UltrapPure 0.5M EDTA pH 8.0Invitrogen - Thermo FisherCatalog #15575020
Trypan Blue Solution 0.4%Thermo Fisher ScientificCatalog #15250061
Consumables:
- Vacutainer Cell Preparation Tubes (CPT) with sodium heparin (BD, Cat.no. 362753)
- Cryovial 1.8 mL Internal Thread PP *VS* (Nunc (Fisher Scientific), Cat. no. NNC368632-PK)
- Pipette Graduated 3ml Sterile Pastette®Alpha LaboratoriesCatalog #LW4112
- Cell Counting Slides for TC10™/TC20™ Cell Counter Dual-Chamber 5 x 30 slides 300 counts #1450015Bio-rad LaboratoriesCatalog #1450015
- CoolCell LX Freezing Container, 12 x 1-2ml cryo vials, Purple (Biocision, Cat. no. BCS-405)
| A | B | |
| Wash Buffer composition (1% FBS, 1 mM EDTA), store at 4°C. | ||
| PBS, pH 7.4 (Gibco, Cat. no. 10010049) | 500 mL | |
| Fetal Bovine Serum (FBS) (Sigma, Cat. no. F2442) | 5 mL | |
| UltraPure 0.5 M EDTA, pH 8.0 | 1 mL |
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Excessive centrifuge speed (over 2000 RCF) may cause tube breakage and exposure to blood and possible injury.
Before start
- The BD Vacutainer® CPT™ Tube (Cat. no.362753) should be at Room temperature (18-25ºC) and properly labeled for patient identification.
- After blood collection, the CPT tube should be stored upright at Room temperature (18-25ºC) until centrifugation. Blood samples should ideally be centrifuged within two hours of blood collection for best results.
PBMCs isolation from CPT™ tube
PBMCs isolation from CPT™ tube
30m
30m
Mix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times.
Centrifuge the CPT tube at 1500 rcf (Relative Centrifugal Force) in a horizontal rotor (swing-out head) for 00:30:00 at 20 °C (Speed change of accel/decel: Soft).
30m
Note
After centrifugation, PBMCs will be in a whitish layer just under the plasma layer.
Using a Pasteur pipette, aspirate approximately half of the plasma without disturbing the PBMC cell layer.
Collect cell layer by pouring and transferring cell layer to a 50 mL size conical centrifuge tube with cap.
Note
Collection of cells immediately following centrifugation will yield best results.
Spin down the collected mixture at 300 rcf for 00:15:00 at 20 °C .
Note
Speed change of accel/decel: Soft. Use Pasteur pipette to remove as much supernatant as possible without disturbing cell pellet.
15m
Using a 5-mL serological pipette, gently resuspend the cell pellet with 3 mL of ACK lysing buffer and incubate for 00:03:00 at Room temperature .
3m
First wash: Add wash buffer to bring volume to 50 mL . Cap tube. Mix cells by inverting tube 5 times.
Centrifuge at 300 rcf (accel/decel: Soft) for 00:15:00 at 20 °C .
15m
Aspirate as much supernatant as possible without disturbing cell pellet.
Second wash: Add wash buffer to bring volume to 20 mL . Cap tube. Mix cells by inverting tube 5 times.
Centrifuge at 300 rcf (accel/decel: Soft) for 00:15:00 at 20 °C .
15m
Aspirate as much supernatant as possible without disturbing cell pellet.
Re-suspend cell pellet in an appropriate volume of wash buffer to bring to a concentration of ~1×106 cells/mL for counting.
Cell counting:
Mix 10 µL of cell suspension with 10 µL of trypan blue.
Apply 10 µL of the mixture to a counting slide.
Count the cells using an automated cell counter within 00:05:00 (concentration tends to range from 5×104 to 1×107 cells/mL).
5m
Centrifuge the remaining suspension at 300 rcf (accel/decel: Soft) for 00:10:00 at 20 °C .
10m
Aspirate as much supernatant as possible without disturbing cell pellet.
Resuspend cell pellet in 1 mL of cold CryoStor CS10 in cryotubes and aliquot into two cryotubes per sample (0.5 mL X 2).
Store the cryotubes into CoolCell LX Freezing Container in a -80 °C freezer Overnight before permanent storage in liquid nitrogen.
10m