TITLE: LymphocyteCollection
AUTHORS: Michaela McCown, Josie A Tueller, Scott Weber, Samuel H Payne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Purpose: To isolate in separate wells of B lymphocytes and wells of T lymphocytes from fresh whole blood. </div><div class = "text-block">Generally, lyse red blood cells and decant to obtain white blood cells. Then, stain with fluorescent antibodies to guide the FACS sort by cell type.</div></div>

[STEPS]
?. [Setup]
Clean biosafety cabinent by UV for 15 minutes, then wipe with Ethanol.Gather pipettes, tubes, racks, and waste container into the cabinent. One person can set up while another is bringing the blood over on ice if two people are available for prep, otherwise leave blood on ice during initial setup.Old blood (such as from a previous draw) was used for PI+ control, however cells may be otherwise lysed for it.
Human blood requires BioSafety Level 2. We used this protocol with human blood from healthy donors collected from the Student Health Center. Alternatives such as mice blood will have different safety requirements.
?. [Setup]
For each control and sample, you will need about  and Round up when aliquoting from the stock.Let both stand at room temperature.We have 10X RBC Lysis Buffer. Prepare the buffer by diluting to 1X with DI Water. For our typical prep of 3 reps x 2 subjects + 4 controls, or 10 tubes, you'll need 25-30 mL PBS and 40-50 mL 1X RBC Lysis Buffer.
2.4 mL
4 mL
?. [Setup]
Prepare 5ml FACS tubes filled with of  at room temp.   1 FACS tube for each replicate - generally 3 per sample  4 more FACS tubes for Controls: Cells Only, FITC only, APC only, PI only (Additional single color controls as necessary.)
[(~4ml)]
?. [Setup]
Vortex the blood if necessary.Add blood samples to the corresponding tube: of blood for each sample of blood for each control    For PI control, use old blood if available; otherwise you will need to lyse or damage the cells
Human blood is handled at BioSafety Level 2.
200 µl
100 µl
?. [Setup]
Wait  for RBC lysis and then centrifuge .
Centrifuge: 5 33, 5 min
?. [Setup]
Decant into waste bleach solution, blot on paper towel without resuspending.If red blood cells are still visible in the pellet, add an additional  of and repeat incubation and centrifugation. Otherwise, the pellet should be white or clear and will be thin.
[(~4ml)]
?. [Lymphocyte staining]
Prepare staining mix in DARK: (cabinet lights off is sufficient)    Master Mix - Per sample: of PBS        Add  CD3 FITC CD19 APC and PI    For single color controls, it may be easiest to add  PBS followed by the stain directly to the FACS tube, rather than mixing the stain in a separate container first.
100 µl
5 µl
5 µl
10 µl
100 µl
?. [Lymphocyte staining]
Add   PBS/stain mix to each sample.
100 µl
?. [Lymphocyte staining]
Incubate cells for  to 20 minutes in the dark, such as covered with tin foil.
?. [Lymphocyte staining]
Add ~  of PBS to each tube, centrifuge
2 mL
Centrifuge: 250 34, 5 min
?. [Lymphocyte staining]
Decant by dump-tap.Resuspend in  PBS.
300 µl
?. [FACS prep]
Filter using 40ul filter prior to any FACS sort. – The cap filters need to be centrifuged, just a minute or two and softly will work.
Centrifuge: 100 34, 1 min
?. [Cleanup]
Wipe the cabinent with ethanol, then turn UV on for It may be helpful to turn it on, take the cells for the sort, then come back or have someone else turn it off.