TITLE: MGH COVID-19 Effort Blood Processing Protocol for PBMC, Neutrophils and Plasma Isolation
AUTHORS: Moshe Sade-Feldman,  Miguel Reyes, Alexandra-Chloe Villani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps and methods for blood processing for PBMC to assist the MGH COVID-19 effort.</div></div>

[STEPS]
?. [PBMC Isolation]
Follow the steps detailed in the "Before Start" section prior to moving forward in this protocol.
?. [PBMC Isolation]
Invert vacutainer 5x to mix blood.
Do not shake.
?. [PBMC Isolation]
For transferring blood to the different tubes, proceed ONLY one patient sample set at a time and avoid opening and closing vacutainers.
?. [PBMC Isolation]
Gently open one vacutainer and move whole blood to a separate 15mL conical tube for neutrophil isolation based on the amount of blood received.4-8ml blood - take 5000ul for neutrophil isolation2-3ml blood - take 250ul for neutrophil isolationdon't take blood for neutrophil isolation if there is
Do not put on ice.
This step could be skipped if neutrophil isolation is not needed.
?. [PBMC Isolation]
Using a 10mL serological pipet, transfer the remaining blood in the EMPTY 50mL tube.
Take note of the total colume of blood transfer (you need the number to calculate RPMI volume).
?. [PBMC Isolation]
After completing blood transfer for neutrophil, disinfect the 15mL conical tubes with 10% bleach solution and handoff the tubes to person #2 who can move the tubes to another BSC to proceed with RBC lysis and neutrophil isolation.
?. [PBMC Isolation]
After finishing the blood transfer, move serological pipet to the bleach bucket.
?. [PBMC Isolation]
For each of the tubes listed in Step 5, make sure to fill with the correct buffer:Fill 50mL conical with (2x volume of blood) RPMI at Fill one 50mL SepMate tube with  and add through the hole in the middle of the cone inside the tube.If the blood volume is
0 Room temperature
[room temperature Ficoll]
[Ficoll]
?. [PBMC Isolation]
Repeat Steps 2-7 until all blood samples have been transferred to the empty 15mL falcon tube and 50mL falcon tube containing blood.
?. [PBMC Isolation]
Mix all the 50mL tubes by inverting 5x.
?. [PBMC Isolation]
Using a different 25mL serological pipet for each patient, transfer the diluted blood into the SepMate tube.
?. [PBMC Isolation]
Slowly pipet down the side of the tube to avoid blood mixing with Ficoll that may come out of the hole in the insert (
?. [PBMC Isolation]
Centrifuge at  with maximum acceleration and the brake on.
Centrifuge: 1200 33, 20 min, 20 10
?. [PBMC Isolation]
During centrifugation, prepare the following:Grab Mr. Frosty from  freezer if not already in lab refrigeratorAn ice bucketPre-fill round-bottom well 96-well plate with   and prepare one well per sample (for counting)Label one 15mL conical for PBMC per sample, leave emptyPre-fill round-bottom well 96-well plate with  and prepare one well per sample (for counting)Grab RPMI bottle from  fridge and keep
-80 °C
[Trypan Blue]
4 °C
on ice
?. [PBMC Isolation]
After centrifugation, carefully remove the SepMate tube and verify the PBMC layer is visible.
If no layer is visible, centrifuge again at   with the brake on.
?. [PBMC Isolation]
Place the SepMate tube into a rack.
?. [PBMC Isolation]
Change the centrifuge temperature to  and close the lid.
4 °C
?. [PBMC Isolation]
Using a 10mL serological pipet, collect  from the tube and transfer into the empty 15mL conical tube.
[plasma]
?. [PBMC Isolation]
Place  until later.
on ice
?. [PBMC Isolation]
Remove remaining plasma by aspirating and leave approximately  above the layer.
3 mL
?. [PBMC Isolation]
With a transfer pipet, collect and transfer PBMC layer from the SepMate tube and move to the 15mL conical tube.
?. [PBMC Isolation]
Top up the conical tube to 15mL with cold RPMI.
?. [PBMC Isolation]
Centrifuge  using max acceleration and max break.
Centrifuge: 300 33, 5 min, 4 10
?. [PBMC Isolation]
Remove the supernatant by aspirating, making sure to avoid disturbing the pellet, and place tube .
on ice
?. [PBMC Isolation]
Resuspend the pellet in .
[cold RPMI]
?. [PBMC Isolation]
Place .
on ice
If pellet is not visible, resuspend in
?. [PBMC Isolation]
Add  to the roung bottom 96-well plate containing .
[cell suspension]
[Trypan Blue]
?. [PBMC Isolation]
Count the cells to determine the yield and number of aliquots to be frozen.
See the "Counting PBMCs" protocol.
?. [PBMC Isolation]
Top up the tube to 10mL with RPMI.
?. [PBMC Isolation]
Centrifuge the cell suspension at  with max acceleration and max break.
Centrifuge: 300 33, 8 min, 4 10
?. [PBMC Isolation]
During centrifugation:gather cell freezing materials: cold Crystor (CS10), refrigerated Mr. Frosty, cryo vialsprint cryotube labels and stick on tubes: lable with "Blood PBMC", subject ID, and timepoint "PXX-DY", aliquot number, number of cells, and datefor each patient, we will freeze aliquots of  cells each and freeze the extra leftover as a spearate aliquot.
If the leftover cell is less than 200K cells, then distribute the 200K cells across all aliquots.
?. [PBMC Isolation]
Remove supernatant through aspiration.
?. [PBMC Isolation]
Resuspend pellet in cold Cryostor (0.5mL per 0.5M cell aliquot) using the total volume to be used to cover all aliquots accounted for per sample.
?. [PBMC Isolation]
Resuspend pellet by gently pipetting up and down 10x.
The Cryostor solution is viscous so be sure to pipette up and down slowly.
?. [PBMC Isolation]
Pipet  intro cryotubes.
[cell suspension]
?. [PBMC Isolation]
Transfer to Mr. Frosty and immediately place in  freezer for a maximum of .
-80 °C
?. [PBMC Isolation]
Record the time the blood sample was frozen.
?. [PBMC Isolation]
The next day, cells can be moved from Mr. Frosty to a cardboard box (labeled PBMC-1...so on).
Note down on the spreadsheet which box and position the samples are placed.
Frozen cells can be kept between   and   in   before being send to the Broad Institute on dry ice.
?. [Counting PBMCs and Determining # of aliquots of PBMCs]
Make sure the cell suspension is well mixed by pietting up and down.
?. [Counting PBMCs and Determining # of aliquots of PBMCs]
Transfer  into cryovial containing  and mix with a pipet slowly, up and down 5x.
[cell suspension]
[Trypan Blue]
?. [Counting PBMCs and Determining # of aliquots of PBMCs]
Load  into one of the hemocytometer reservoirs.
[diluted cells]
If using an automated cell counter(Countess- Invitrogen or BioRad), make sure to load 10ul of the diluted cells into the right plastic cell counter chamber. Insert into the machine and press the "Count Cells" button. Cells will be counted automatically and concentration per ml will be given at the end, including the viability of cells.When using the BioRad counting machine, make sure the lower gate is on 7um to avoid counting red blood cells.
?. [Counting PBMCs and Determining # of aliquots of PBMCs]
Count the number of cells in one corner 4x4 grid (count all cells in the squares).PBMC cells should have a light blue hue (due to nucleus staining)count the cells in contact with the borders of only two sides of the squares
?. [Plasma Protocol]
Once samples are in the freezer, centrifuge the plasma at  with max acceleration and max brake.
Centrifuge: 1000 33, 5 min, 4 10
?. [Plasma Protocol]
During centrifugation, print cryotube labels an stick on 3 cryotubes: label with "Plasma", subject ID, timepoint "PXX-DY", aliquot number, and date.
?. [Plasma Protocol]
Without disturbing the pellet, aliquot  into 3 cryovials (4.5mL total).
[supernatant]
The remaining can be discarded.
?. [Plasma Protocol]
Freeze the aliquoted plasma at  in a normal cardboard box or plastic box (labeled Plasma-1...so on).
-80 °C
?. [Plasma Protocol]
Take note on the spreadsheet which box and position the samples are placed.
?. [Plasma Protocol]
Take note of the time of when the samples were frozen.
Plasma samples can be shipped to the Broad Institute with the PBMCs.
?. [Neutrophil Isolation (OPTIONAL)]
Before starting the upcoming sections:Set centrifuge to room temperature for RBC lysisHave EasySep magnet on handIf the anticoagulant in the vacuatiner is not EDTA, add 1uL of 0.5M EDTA to the blood Make PBS+EDTA buffer: 500mL PBS + 1mL of 0.5M EDTA and keep at room temperatureLabel three round-bottom tubes and one 15,L conical tube with patient ID
?. [RBS Lysis]
Organize the 15mL conical tubes containing  in the hood.
This step should be performed while the other person is preparing for Ficoll isolation.
[whole blood]
?. [RBS Lysis]
Add  to blood using a 10Ml serological pipette and mix the sample 5 times.
[ACK lysis buffer]
?. [RBS Lysis]
When done, move the pipette to the bleach bucket.
?. [RBS Lysis]
Spin  with max acceleration and max deceleration.
Centrifuge: 300 34, 5 min
?. [RBS Lysis]
Aspirate supernatant.
Be careful not to disturb the pellet.
?. [RBS Lysis]
Resuspend cells in  at  by pipetting up and down gently 5x.
[PBS+EDTA buffer]
0 Room temperature
?. [RBS Lysis]
Continue to EasySep isolation.
?. [Neutrophil EasySep Isolation]
Vortex RapidSpheres for .
Aft anytime, DO NOT cap the 15mL tube.
?. [Neutrophil EasySep Isolation]
Mix the isolation cocktail with 200uL pipette up and down 10x.  ABC1RapidSphere volumeIsolation Cocktail volume2500-1mL blood50uL50uL3250uL blood25uL25uL
ABC1RapidSphere volumeIsolation Cocktail volume2500-1mL blood50uL50uL3250uL blood25uL25uL
?. [Neutrophil EasySep Isolation]
Add isolation cocktail to sample.
?. [Neutrophil EasySep Isolation]
Add RapidSpheres to sample.
?. [Neutrophil EasySep Isolation]
Mix gently by pipetting up and down 10x.
?. [Neutrophil EasySep Isolation]
Incubate for  at .
0 Room temperature
?. [Neutrophil EasySep Isolation]
After , top up to  with PBS+EDTA buffer at .
4 mL
0 Room temperature
?. [Neutrophil EasySep Isolation]
Close the tube tightly and then mix gently by tilting the tube back and forth 5x.
?. [Neutrophil EasySep Isolation]
Place tube into magnet.
Make sure each 15mL tube sits well at the bottm of it without lid.
?. [Neutrophil EasySep Isolation]
Incubate at  for   on magnet.
0 Room temperature
While on the magnet, label a second set of 15mL tubes.
?. [Neutrophil EasySep Isolation]
After , using a 5mL serological pipet, transfer supernatant into new 15mL tube (tube 2).
Angle the pipet such that the tip is opporsite the magnet to avoid toughing the beads.
Procedure is negative selection so neutrophils will be in supernatant.
?. [Neutrophil EasySep Isolation]
Add  to the new 15mL tube (tube 2) if started with  or if started with
[RapidSpheres]
[whole blood]
12.5 µl
[whole blood.]
?. [Neutrophil EasySep Isolation]
Close the tube tightly and mix gentle by tilting the tube back and forth 5x (avoid bubbles.
?. [Neutrophil EasySep Isolation]
Incubate  at .
0 Room temperature
?. [Neutrophil EasySep Isolation]
After , place tube on magnet (without lid).
?. [Neutrophil EasySep Isolation]
Incubate  at  on magnet.
0 Room temperature
While on magnet, label a third set of 15mL tubes.
?. [Neutrophil EasySep Isolation]
After , using a 5mL serological pipet, transfer supernatant into new 15mL tube (tube 3).
?. [Neutrophil EasySep Isolation]
Place the new tube (tube 3) with the supernatant back on th magnet.
?. [Neutrophil EasySep Isolation]
Incubate for  at  on magnet.
0 Room temperature
While on magnet, label a 15mL flacon tube (tube 4).
?. [Neutrophil EasySep Isolation]
After , using a 5mL serological pipet, transfer supernatant (~3.5mL) into 15mL tube (tube 4).
?. [Neutrophil EasySep Isolation]
Spin  with full acceleration and full deceleration.
Centrifuge: 300 33, 5 min
?. [Neutrophil EasySep Isolation]
Aspirate supernatant, being careful not to disturb the pellet.
?. [Neutrophil EasySep Isolation]
Resuspend in  and mix well.
[PBS+EDTA]
?. [Neutrophil EasySep Isolation]
Count the cells to determine the yield and number of aliquots to be frozen.
See "Cell Counting" in the next section.
?. [Cell Counting]
Add  to .
[resuspended cells]
[Trypan Blue]
?. [Cell Counting]
Mix well by pipeting up and down.
?. [Cell Counting]
Add  to a hemocytometer.
10 µl
If using an automated cell counter(Countess- Invitrogen or BioRad), make sure to load 10ul of the diluted cells into the right plastic cell counter chamber. Insert into the machine and press the "Count Cells" button. Cells will be counted automatically and concentration per ml will be given at the end, including the viability of cells.When using the BioRad counting machine, make sure the lower gate is on 4um to avoid counting red blood cells.
?. [Cell Counting]
Count the number of cells with a hemocytometer.There are four section of 4x4 grids (outlined in red below)Count cells in two of the 4x4 grids (take average between the two grid counts)When counting, include cells on the edges of 2 sides of each grid, and exclude cells on the edges of the other 2 sides of each grid (as shown below on right)If there are not many cells, count number of cells in all four grids and take the average of all four (will be more accurate)Take average of cells counted in 4x4 grids to use as "# cells" for the calculation below.
?. [Cell Lysis]
Desired outcome:2 cryotubes per patient, each tube with 100uL of neutrophil lysate in eachfinal concentration of cells in RNA lysis buffer = 1000 cells per ula total of 200K cells to split between two cryotubes
?. [Cell Lysis]
Mix 200K cells to a 1.5mL Eppendorf tube by transferring an appropriate volume of the cell suspension.
?. [Cell Lysis]
Determine fraction of cells to transfer to new tube: 20/('# cells").
?. [Cell Lysis]
Top up tube of cells to  with PBS+EDTA.
1.5 mL
?. [Cell Lysis]
Spin .
Centrifuge: 300 33, 5 min
?. [Cell Lysis]
Make fresh RNA lysis buffer (TCL + 1% bME) according to number of samples received.
Make sure to RNAZap the surface of the hood where the lysis buffer will be used and pipettes to be used and use Rnase free tips for pipetting.
?. [Cell Lysis]
Print cryotube labels and stick on tubes: label with "Neu Lysate", subject ID and timepoint "PXX-DY", aliquot number, concentration of cells ("1k/uL"), and date.
?. [Cell Lysis]
Pipe out supernatant and remove excess with P200 (be cautious).
?. [Cell Lysis]
Resuspend in .
[Lysis Buffer]
If lower number of cells are received, resuspend cells in lower volumes to reach 1000 cell/1mL.
?. [Cell Lysis]
If less than 10,000 cells are recovered, resuspend in  and note cell concentration on the cryotube. Freeze single cryotube.
10 µl
?. [Cell Lysis]
After resuspending cells in , mix well and transfer  to two cryotubes.
200 µl
100 µl
?. [Cell Lysis]
Place tubes on dry ice immediately.
?. [Cell Lysis]
After , the aliquoted lysates can be moved to the  in a normal cardboard or plastic box.
-80 °C
Note down on the spreadsheet which box and position the samples are placed and take note of the time when the samples were frozen.