Aug 16, 2023
Preparation and cryopreservation of human whole blood samples for analysis by flow cytometry (fresh or after cryobanking)
- 1Charité - Universitätsmedizin Berlin
Protocol Citation: Wiebke Werner, Linda Hammerich 2023. Preparation and cryopreservation of human whole blood samples for analysis by flow cytometry (fresh or after cryobanking). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d91zg3e/v1
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 30, 2023
Last Modified: August 16, 2023
Protocol Integer ID: 82622
Keywords: Human Samples, Whole Blood, Flow Cytometry, FACS, Blood, human, immune monitoring, peripheral immune cells, cryopreservation
Abstract
This protocol focuses on the preparation of human whole blood samples for analysis by flow cytometry.
It provides two options for sample preparation: (1) immediate flow cytometry with fresh blood samples or (2) cryopreservation of the samples at -80°C using the Stable-Lyse/Store V2 system from SmartTube Inc. for flow cytometry at a later timepoint.
Note: This protocol does not provide guidelines on how to create a multicolor flow cytometry panel!
Guidelines
- This protocol is only tested on EDTA anti-coagulated whole blood.
- Experiments for whole blood samples are performed at room temperature. Nevertheless, if you cannot process your sample directly after collection, keep the collection tubes at 4 °C until processing, especially if you want to store the plasma for later analysis (however, extended storage periods, ie. several hours, might influence frequency and phenotype of specific immune cell types, especially myeloid cells).
- Work with filter tips!
- When working with fluorochrome-conjugated antibodies, avoid bright (sun) light and consider using aluminum foil to protect your samples from light while incubating.
Advantages:
- Analyze all major immune cell subtypes including granulocytes with flow cytometry.
- No pre-selection/exclusion of certain immune cell subpopulations (e.g. like when isolating PBMC).
- The procedure itself is easier and faster, as it does not require densitiy-gradient centrifugation.
- Ready-to-use quality-controlled cryo-preservation buffers.
Limitations:
- As mentioned above, this protocol has only been tested on EDTA-anticoagulated blood.
Qualifications:
We recommend basic experience in wet-lab work (e.g. how to pipette) to handle this protocol.
Materials
INSTRUCTIONS FOR REAGENT AND BUFFER PREPARATION:
Blocking Buffer:
- transfer 1 gram of BSA to a 50 ml falcon
- add 1 ml of each type of seraum (human, mouse, rabbit, rat)
- add PBS ad 50 ml
- mix until dissolved
- aliquot and store at -20°C
FACS Buffer:
- 500 ml DPBS + 2 mM EDTA (2 ml 0.5M EDTA)
Fixation Buffer (2% PFA in PBS):
- dilute 4% PFA in PBS (1:1)
1X Lysis Buffer:
- 9 ml dH2O + 1 ml BD Pharm Lyse (1:10)
REAGENTS:
Albumin bovine-serumMerck MilliporeSigma (Sigma-Aldrich)Catalog #A4503 (BSA)
1X Dulbecco’s Phosphate Buffered Saline (DPBS) Thermo Fisher ScientificCatalog #14190094
EDTA - Solution pH 8.0Panreac AppliChemCatalog #A3145
BD Pharm Lyse™BD BiosciencesCatalog #555899
4% Paraformaldehyde in PBSAlfa AesarCatalog #J61899-AK
Sera from humanMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2257-1ml
Sera from mouseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S3509-1ml
Normal rabbit serum (invitrogen)Thermo Fisher ScientificCatalog #10510
Normal rabbit serum (invitrogen)Thermo Fisher ScientificCatalog #10710C
+ fluorochrome-conjugated antibodies of your choice
+ fixable viability stain (if desired)
REAGENTS ONLY NEEDED FOR CRYOFIXATION:
Stable-Lyse V2SMART TUBE Inc.Catalog #Stable-Lyse V2
Stable-Store V2SMART TUBE Inc.Catalog #Stable-Store V2
DISPOSABLES OF YOUR CHOICE:
- EDTA blood collection tube
- Cryotubes (1,8 ml)
- Microcentrifuge tubes (1,5 ml)
- Pipet filter tips (10, 200 and 1000 µl)
- 5 ml FACS tubes
- caps for FACS tubes
Equipment:
- biosafety cabinet (BSL2)
- centrifuge
- pipets (0,1-2,5 µl, 0.5-10 µl, 10-100 µl, 100-1000 µl)
- pipette controller
Safety warnings
If you work with untested human samples (e.g. status of Hepatitis B/C and HIV is unknown), all experiments must be carried out in a Class II biosafety cabinet (with laminar air flow). Liquid waste has to be collected and autoclaved before disposal, contaminated materials have to be collected and disposed of separately from other lab waste as they are potentially infectious.
Use caps/lids to close your FACS tube when removing them from the biosafety cabinet for centrifugation. Use pipet tips with filters.
Depending on your personal safety preferences as well as the specific regulations of your research department/institute/country, there might be other or additional regulations to consider. Contact your biosafety officer!
Ethics statement
Before you work with human samples, you have to acquire an human ethics approval from the local ethics committee of your institution! Every subject/patient has to give informed consent!
Before start
- Make sure you read the safety warnings regarding untested human samples!
- Make sure an ethics approval according to your institution´s guidelines has been obtained before working with subjects or patient samples!
- Revisit the materials list to make sure all the equipment, materials and reagents are available to you.
Depending on whether you want to perform flow cytometry on fresh or cryo-preserved samples, you can select different protocol options at this step.
Fresh whole blood (WB) samples for flow cytometry25 steps
Use this protocol if you want to perform flow cytometry immediately after blood sample collection ("fresh").
Preparations
Preparations
30m
30m
Label your 5 ml FACS tubes. You might need several tubes per sample, depending on the number of panels and controls you are planning to run.
Note
GOOD TO KNOW - FMO (fluorescence minus one) controls are helpful to discriminate positive and negative (unspecific background fluorescence) signals, especially if the positive cell populations are not distinctly separated from the negative population. In an FMO tube, all fluorochromes in the panel are present except the fluorochrome in question.
5m
Prepare the antibody mix: For each FACS tube, use 20 µL blocking buffer and the appropriate amount of each of your antibodies. Always prepare the master mix for one extra sample or add 10% for pipetting errors.
Note
EXAMPLE 1
- 2 blood samples (one full stain and one control each) + 1 extra for pipetting error = 5x mix
- 20 antibodies in panel (1µl needed per sample)
- Blocking Buffer: 5 x 20 µl = 100 µl
- 5 µl per antibody
- final volume of antibody master mix: 200 µl
- mix to add per FACS tube = 20 µl blocking buffer + 20 µl antibodies = 40 µl
EXAMPLE 2
- 2 blood samples (one full stain and one control each) = 4x mix +10%
- 20 antibodies in panel (1µl needed per sample)
- Blocking Buffer: 4x 20 µl = 80 µl (+ 10%) = 88 µl
- Each antibody: 4 µl per antibody +10% = 4,4 µl per antibody
- final volume of antibody master mix: 176 µl
- mix to add per FACS tube = 20 µl blocking buffer + 20 µl antibodies = 40 µl
25m
Collect peripheral blood by venipuncture directly into a collection tube with EDTA and invert several times to mix and ensure proper anti-coagulation
Note
Blood samples should be processed as soon as possible. If immediate processing is not possible, keep samples at 4°C.
Staining (BSL2)
Staining (BSL2)
25m
25m
Invert the EDTA blood collection tube several times to homogenize, especially if the samples have been kept in the fridge.
1m
Transfer 200 µL of whole blood to each of your prelabeled 5 ml FACS tubes.
30s
Add the appropriate concentration of fixable viability stain directly to the whole blood in each of the FACS tubes and mix well by flicking the tube or vortexing.
Note
HOW TO "FLICK"- Mix well by holding the upper part of the tube between thumb and index finger of one hand and at the same time, gently flick the bottom of the tube repeatedly with the index finger of the other hand.
1m
Incubate for 00:05:00 at Room temperature .
5m
Add the appropriate amount of the prepared antibody mix (see Step 3) to each tube. Gently pipette up and down to mix.
Note
EXPERT TIP - In case you are using FMO control(s), you can prepare the antibody mix for all your tubes without the FMO antibody(ies) and add them later to your full stain tube.
1m
Incubate 00:20:00 at Room temperature .
20m
Red blood cell lysis (BSL2)
Red blood cell lysis (BSL2)
17m
17m
Add 2 mL 1X Lysis Buffer to each FACS tube and mix well by pipetting up and down.
Note
HOW TO - Works best using a 1000 µl pipet
1m
Incubate for 00:10:00 at Room temperature .
10m
Add 2 mL FACS buffer and mix well by pipetting up and down.
1m
Spin 500 x g, Room temperature, 00:05:00
5m
Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.
Note
HOW TO DECANT - Empty the supernatant with momentum and - while keeping the FACS tube in an upside-down position to not disrupt the pellet on the bottom - dip the tube onto a paper towel to get rid of the excess liquid. Turn the FACS tube upright and gently flick to resuspend.
2m
Add 1 mL 1X Lysis Buffer and mix well by pipetting up and down.
1m
Incubate 00:05:00 at Room temperature .
5m
Add 2 mL FACS buffer and mix well by pipetting up and down.
1m
Spin 500 x g, Room temperature, 00:05:00
5m
Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
2m
Fixation
Fixation
15m
15m
Add 1 mL Fixation Buffer and mix well by pipetting up and down.
1m
Incubate for 00:10:00 at Room temperature .
10m
Add 2 mL FACS buffer and mix well by pipetting up and down.
Note
After fixation, samples can be further processed on the bench, a biosafety cabinet is no longer necessary.
1m
Spin 500 x g, Room temperature, 00:05:00
5m
Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
2m
Add 200 µL FACS buffer and proceed to sample aquisition.
Note
Fixed samples can be stored at 4 °C overnight and recorded the next day. However, keep in mind that fixatives such as formalin can change the autofluorescence of your cells and destabilize fluorochromes, especially tandem dyes. If possible, record you samples directly after fixation.
1m
Protocol references