TITLE: Preparation and cryopreservation of human whole blood samples for analysis by flow cytometry (fresh or after cryobanking)
AUTHORS: Wiebke Werner, Linda Hammerich

[DESCRIPTION]
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!

[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.

[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.


[STEPS]
1. Depending on whether you want to perform flow cytometry on fresh or cryo-preserved samples, you can select different protocol options at this step. 
SECTION: Preparations
2. 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.
 

3. 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.
 

SECTION: Staining (BSL2)
5. Invert the EDTA blood collection tube several times to homogenize, especially if the samples have been kept in the fridge.

SECTION: Staining (BSL2)
6. Transfer 200 µL of whole blood to each of your prelabeled 5 ml FACS tubes. 

SECTION: Staining (BSL2)
7. 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.

 

SECTION: Staining (BSL2)
8. Incubate for 5 min at Room temperature .

SECTION: Staining (BSL2)
9. Add the appropriate amount of the prepared antibody  mix (see Step 3) to each tube. Gently pipette up and down to mix.


 


SECTION: Staining (BSL2)
10. Incubate 20 min at Room temperature .

SECTION: Red blood cell lysis (BSL2)
11. Add 2 mL 1X Lysis Buffer to each FACS tube and mix well by pipetting up and down.

 

SECTION: Red blood cell lysis (BSL2)
12. Incubate for 10 min at Room temperature .

SECTION: Red blood cell lysis (BSL2)
13. Add 2 mL FACS buffer and mix well by pipetting up and down.

SECTION: Red blood cell lysis (BSL2)
14. Spin 500 x g, 5 min 

SECTION: Red blood cell lysis (BSL2)
15. Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.
 

SECTION: Red blood cell lysis (BSL2)
16. Add 1 mL 1X Lysis Buffer and mix well by pipetting up and down.

SECTION: Red blood cell lysis (BSL2)
17. Incubate 5 min at Room temperature.

SECTION: Red blood cell lysis (BSL2)
18. Add 2 mL FACS buffer and mix well by pipetting up and down.
SECTION: Red blood cell lysis (BSL2)
19. Spin 500 x g, 5 min 

SECTION: Red blood cell lysis (BSL2)
20. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Fixation
21. Add 1 mL Fixation Buffer and mix well by pipetting up and down.
SECTION: Fixation
22. Incubate for 10 min at Room temperature.
SECTION: Fixation
23. Add 2 mL FACS buffer and mix well by pipetting up and down.

 

SECTION: Fixation
24. Spin 500 x g, 5 min
SECTION: Fixation
25. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Fixation
26. Add 200 µL FACS buffer and proceed to sample aquisition.
 


SECTION: Fixable Viability Stain (BSL2)
3. Invert the EDTA blood collection tube several times to homogenize, especially if the samples have been kept in the fridge.
SECTION: Fixable Viability Stain
None. 
SECTION: Fixable Viability Stain (BSL2)
3. If you do not need to include a fixable viability stain, add 250 µL whole blood to each of your 1,8 ml cryotubes and directely proceed with   
 

SECTION: Fixable Viability Stain (BSL2)
3.1. In case you want to use a fixable viability stain, add 250 µL whole blood to each of your pre-labeled 5 ml FACS tubes.
SECTION: Cryopreservation of whole blood samples (BSL2)
3. Add 350 µL Stable-Lyse Buffer to each cryotube containing whole blood and mix well by pipetting up and down.

SECTION: Cryopreservation of whole blood samples (BSL2)
3. Incubate 15 min at Room temperature .

SECTION: Cryopreservation of whole blood samples (BSL2)
3. Add 1000 µL Stable-Store Buffer to each cryotube and mix well by pipetting up and down. 

SECTION: Cryopreservation of whole blood samples (BSL2)
3. Close the cryotubes and incubate 15 min at Room temperature .

 


SECTION: Cryopreservation of whole blood samples (BSL2)
3. Immediately transfer to -80 °C for long-term storage. Samples can be stored for several months.
SECTION: Fixable Viability Stain (BSL2)
2.1. Add the appropriate amount of fixable viability stain directly to the whole blood samples and mix well by flicking the tube. (If you are including an unstained control, do NOT add fixable viability stain to this tube).

 

SECTION: Fixable Viability Stain (BSL2)
2.2. Incubate 5 min at Room temperature .

SECTION: Fixable Viability Stain (BSL2)
2.3. After incubation, add 2 mL FACS buffer to each tube and mix well by pipetting up and down.

 


 


SECTION: Fixable Viability Stain (BSL2)
2.4. Spin 500 x g, 5 min .

SECTION: Fixable Viability Stain (BSL2)
2.5. Using a 1000 µl pipet, carefully pipette off 2 mL of the supernatant without disrupting the cells (including erythrocytes) on the bottom of the tubes.

SECTION: Fixable Viability Stain (BSL2)
2.6. Gently mix the remaining volume (it should be approximately 250 µL ) and transfer to the corresponding 1,8 ml cryotube.

SECTION: Prepare samples for staining
3. Add 2 mL FACS buffer to each tube and mix well by pipetting up and down.

SECTION: Prepare samples for staining
3. Spin 500 x g, 5 min .

SECTION: Prepare samples for staining
3. Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.
 

SECTION: Prepare samples for staining
3.   and repeat wash-spin-discard supernatant (steps 13-15). Afterwards, follow-up with step 16.

SECTION: Prepare samples for staining
3. In the meantime, prepare the antibody mix: For each FACS tube, use 20 µL  blocking buffer and the appropriate amount of each of your antibodies. Count 1 extra sample or 10% for pipetting errors.

 

SECTION: Prepare samples for staining
3. Transfer your samples from -80 °C  to an open rack in the fridge (4 °C ) and let them thaw for approximately 60 min .

SECTION: Staining
3. Add the appropriate amount  of the prepared antibody  mix to each tube. Gently pipette up and down to mix.

 

SECTION: Staining
3. Incubate 20 min at Room temperature .

SECTION: Staining
3. Add 2 mL FACS buffer to each tube.

SECTION: Staining
3. Spin 500 x g, 5 min 

SECTION: Staining
3. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Staining
3. Add 200 µL FACS buffer and proceed to sample aquisition.

SECTION: Prepare samples for staining
3. Transfer the thawed samples to their corresponding 5 ml FACS tubes.
4. Collect peripheral blood by venipuncture directly into a collection tube with EDTA and invert several times to mix and ensure proper anti-coagulation

 

None. 
None. 
None. 
None. 
2. 
SECTION: Preparations
2. Label 5 ml FACS tubes and 1,8 ml cryotubes to store your samples. Remember to prepare additional aliquots to allow for staining controls at analysis (such as FMO or unstained control). 
 
add step for venipunture here!!
SECTION: Fixable Viability Stain (BSL2)
4. Invert the EDTA blood collection tube several times to homogenize, especially if the samples have been kept in the fridge.
SECTION: Preparations
3. Collect peripheral blood by venipuncture directly into a collection tube with EDTA and invert several times to mix and ensure proper anti-coagulation

 

SECTION: Fixable Viability Stain (BSL2)
5. If you do not need to include a fixable viability stain, add 250 µL whole blood to each of your 1,8 ml cryotubes and directely proceed with   
 

SECTION: Fixable Viability Stain (BSL2)
5.1. In case you want to use a fixable viability stain, add 250 µL whole blood to each of your pre-labeled 5 ml FACS tubes.
SECTION: Fixable Viability Stain (BSL2)
5.2. Add the appropriate amount of fixable viability stain directly to the whole blood samples and mix well by flicking the tube. (If you are including an unstained control, do NOT add fixable viability stain to this tube).

 

SECTION: Fixable Viability Stain (BSL2)
5.3. Incubate 5 min at Room temperature .

SECTION: Fixable Viability Stain (BSL2)
5.4. After incubation, add 2 mL FACS buffer to each tube and mix well by pipetting up and down.

 


 


SECTION: Fixable Viability Stain (BSL2)
5.5. Spin 500 x g, 5 min .

SECTION: Fixable Viability Stain (BSL2)
5.6. Using a 1000 µl pipet, carefully pipette off 2 mL of the supernatant without disrupting the cells (including erythrocytes) on the bottom of the tubes.

SECTION: Fixable Viability Stain (BSL2)
5.7. Gently mix the remaining volume (it should be approximately 250 µL ) and transfer to the corresponding 1,8 ml cryotube.

SECTION: Cryopreservation of whole blood samples (BSL2)
6. Add 350 µL Stable-Lyse Buffer to each cryotube containing whole blood and mix well by pipetting up and down.

SECTION: Cryopreservation of whole blood samples (BSL2)
7. Incubate 15 min at Room temperature .

SECTION: Cryopreservation of whole blood samples (BSL2)
8. Add 1000 µL Stable-Store Buffer to each cryotube and mix well by pipetting up and down. 

SECTION: Cryopreservation of whole blood samples (BSL2)
9. Close the cryotubes and incubate 15 min at Room temperature .

 


SECTION: Cryopreservation of whole blood samples (BSL2)
10. Immediately transfer to -80 °C for long-term storage. Samples can be stored for several months.
SECTION: Prepare samples for staining
11. Transfer your samples from -80 °C  to an open rack in the fridge (4 °C ) and let them thaw for approximately 60 min .

SECTION: Prepare samples for staining
12. In the meantime, 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.

 

SECTION: Prepare samples for staining
13. Transfer the thawed samples to their corresponding 5 ml FACS tubes.
SECTION: Prepare samples for staining
14. Add 2 mL FACS buffer to each tube and mix well by pipetting up and down.

SECTION: Prepare samples for staining
15. Spin 500 x g, 5 min .

SECTION: Prepare samples for staining
16. Discard the supernatant by decanting and resuspend the cell pellet in the remaining buffer by flicking the tube.
 

SECTION: Prepare samples for staining
17.   and repeat wash-spin-discard supernatant (steps 13-15). Afterwards, follow-up with step 18.

SECTION: Staining
18. Add the appropriate amount  of the prepared antibody  mix to each tube. Gently pipette up and down to mix.

 

SECTION: Staining
19. Incubate 20 min at Room temperature .

SECTION: Staining
20. Add 2 mL FACS buffer to each tube.

SECTION: Staining
21. Spin 500 x g, 5 min 

SECTION: Staining
22. Discard the supernatant and resuspend the cell pellet in the remaining buffer by flicking the tube.
SECTION: Staining
23. Add 200 µL FACS buffer and proceed to sample aquisition.