αCD45 infusions

Alexa Fluor – antibody conjugations

Reagents:

Bicarbonate reaction buffer 

DMSO (anhydrous)

Alexa Fluor (NHS ester) – 1 mg/tube

Unconjugated antibody

Tris-NaCl storage buffer

Materials:

1.5 mL amber Eppendorf tubes

Rotator

15 mL conical tubes

2 mL Zeba columns

• Note: you can use other sized columns based on the amount (volume) of antibody you want to conjugate. Change wash volumes, spin speeds, and times according to product specifications.
• Note: the maximum antibody concentration we have tested for the columns is 10 mg/mL and that works fine. 

Preparation:

1. Make bicarbonate buffer (pH 9.5) - 

    To make 1 L:

    17 g Na₂CO₃

    28g NaHCO₃

    pH to 9.5
 

2. Make Tris-NaCl storage buffer (pH 8.2) – 10 mM Tris, 150 mM NaCl

    To make 1 L: 

    1.42 g TRIZMA 8.0

    8.77 g NaCl

    pH to 8.2 

    *if antibodies will not be infused into animals, you can add 0.1% NaN₃ to the storage buffer

*I have made these buffers and kept them at RT for more than a year without any problems. 

Conjugation:

1. Bring dessicated fluor to room temperature.
   • Note: fluors should be stored dry and protected from light at -20 C. 
2. Prepare Zeba column with bicarb buffer.
   1. Break off stopper, put column in 15 mL conical, and spin once at 1000 x g for 2 minutes to remove storage buffer. Aspirate flowthrough. 
   2. Add 1 mL bicarb buffer, spin at 1000 x g for 2 minutes, aspirate flowthrough.
   3. Add 1 mL bicarb buffer, spin at 1000 x g for 2 minutes, and aspirate flowthrough.
   4. Add 1 mL bicarb buffer, spin at 1000 x g for 3 minutes, and aspirate flowthrough.
3. Add 1 mL of unconjugated antibody to prepared column. Spin at 1000 x g for 3 minutes and transfer antibody to an amber tube.
4. Resuspend the fluor with 100 uL anhydrous DMSO to get a concentration of 10 mg/mL. Pipette up and down and/or vortex to fully mix. 
5. Add dye to antibody in a 10:1 molar ratio. 

• Alexafluor molecular weights can be found on the ThermoFisher website and I’ve included the MW of the dyes I generally use at the bottom. 
• For example, if you wanted to conjugate 1 mg of antibody to Alexa Fluor 488 NHS Ester…
  • You have 1 mg of antibody with a MW of 150,000 g/mol so you have 6.67 nmol of antibody to conjugate.
  • For a 10:1 molar ratio of dye:antibody, you’ll need to add 66.7 nmol of dye.
  • The MW of Alexa Fluor 488 is 643.4 g/mol and you want 66.7 nmol so you need 42.9 ug of dye.
  • The dye has been resuspended to a concentration of 10 mg/mL so you need 4.29 uL of dye to get 42.9 ug. 

6. Flick to mix and place on rotator (at room temperature, protected from light) for 2 hours.

7. Prepare 2 mL Zeba column with Tris-NaCl storage buffer.

1. Break off stopper, put column in 15 mL conical tube, and spin once at 1000 x g for 2 minutes. Aspirate flowthrough.
2. Add 1 mL Tris-NaCl buffer, spin at 1000 x g for 2 minutes, and aspirate flowthrough.
3. Add 1 mL Tris-NaCl buffer, spin at 1000 x g for 2 minutes, and aspirate flowthrough.
4. Add 1 mL Tris-NaCl buffer, spin at 1000 x g for 3 minutes, and aspirate flowthrough. Discard 15 mL conical tube. 

8. Get a new 15 mL conical tube for conjugated antibody collection. 

9. Briefly spin down amber tubes (to get any liquid from the lid) and add all solution to the Tris-NaCl-prepared column. Spin at 1000 x g for 3 minutes. 

10. Collect flowthrough and transfer to light protected tube. Measure concentration and titrate on cells before use. 

Alexa Fluor MW:

Analysis of Lymph Node Trafficking by Serial Intravascular Staining

Materials

• Rhesus macaques: Indian origin, <10 kg, male or female
• HBSS: Gibco, calcium, magnesium, no phenol red (ThermoFisher Scientific catalog 14025134)
• Antibodies for intravascular infusion: anti-CD45 clone ITS_rhCD45 conjugated to Alexafluor dyes, available from the Roederer Lab and the NHP reagent resource.  Note: biotin conjugates perform very poorly and should not be used.  We have not tested other hapten conjugates.
• LIVE/DEAD fixable blue dead cell stain kit (ThermoFisher Scientific catalog L23105) 
• Antibodies for ex vivo staining:
  • Note: we recommend including anti-CD45 clone D058-1283 (BD Biosciences)
  • Note: use Super Bright Complete Staining Buffer, if needed. Invitrogen eBioscience (ThermoFisher Scientific catalog SB-4401-75)
• RPMI 1640 media: Gibco, no glutamine (ThermoFisher Scientific catalog 21870076)
• Fetal bovine serum: Gibco, heat inactivated (ThermoFisher Scientific catalog 10438026)
• Penicillin-Streptomycin: Gibco, 5,000 U/mL (ThermoFisher Scientific catalog 15070063)
• HEPES buffer: 25 mM HEPES with 113 mM sodium chloride
• Newborn calf serum: (HINCS) R&D Systems, heat inactivated (R&D systems catalog S11250H)
• PBS: Gibco 1x DPBS, no calcium, no magnesium (FisherScientific catalog 14-190-250)
• Ketamine: Zetamin (VET-ONE)
• Xylazine: ANASED INJECTION (Akorn Incorporated)
• Paraformaldehyde (20%)
• Cell freezing media: CryoStor CS10 (Millipore Sigma catalog C2874-100ML)

Equipment

• gentleMACS C tubes (Miltenyi Biotec catalog 130-093-237)
• 50 mL conical centrifuge tubes: Falcon (Fisher Scientific 14-432-22) 
• 70 µM cell strainer (Fisher Scientific catalog 08-771-2) 
• gentleMACS dissociator (Miltenyi Biotec catalog 130-093-235)
• Syringes: Covidien Monoject 6 mL luer-lock top (ThermoFisher Scientific catalog 05-561-65)
• Needles: various sizes (Monoject)
• Catheter: various sizes (Terumo)
• Scalpels: Andwin Scientific feather disposable scalpel #21(ThermoFisher Scientific catalog NC0595256)
• Petri dishes: Corning, 150 x 15 mm (VWR catalog 25373-187)
• Forceps: Fisherbrand high precision straight broad/strong point forceps (ThermoFisher catalog 12-000-128)
• Cryovials: Nunc, 1.8 mL (ThermoFisher catalog 377267)
• FACS tubes: Corning, 12 x 75 mm, 5 mL (VWR catalog 60819-138)

Prep:

1. Make R10 buffer – Add 10% HIFBS and 1% pen-strep to RPMI 1640. 
   • Keep at 4 C when not in use. 
   • Buffer may be used for ~1 month. 
2. Make staining buffer - add 4% HINCS to 25 mM HEPES buffer with 113 mM sodium chloride

Animal facility:

1. Sedate animals with ketamine (10 mg/kg body weight (BW), intramuscularly (I/M) or subcutaneously (S/Q) and Xylazine (0.5 mg/kg BW, I/M or S/Q) prior to inoculations and blood withdrawals.
2. Prepare antibody for infusion.  Dilute mAb in sterile 1x HBSS to 30 ug/kg body weight (BW), diluted to an injection volume of 5 mL. Protect fluorescently-conjugated mAbs from light. Transfer diluted antibody into a syringe with needle. 
3. For single infusion experiments, intravenously inject 5 mL antibody into saphenous vein slowly (over ~30 seconds). Euthanize the animal after 5 minutes. Sample blood and harvest tissue after euthanasia.  
4. For SIVS experiments with multiple infusions,, place a catheter in the saphenous vein for infusions. Serial intravascular infusions should be given slowly (over ~30 seconds). Blood should be sampled from a distal site relative from the infusion site 5 minutes after each infusion. 

• We have performed up to 5 infusions over the course of 7 days with at least 55 minutes between individual infusions. 
• If the animal is to be necropsied, the final infusion should be given 5 minutes before euthanasia. 

5. Collect tissues of interest and store in R10 on ice. Protect samples infused with fluorescently-conjugated antibody from light. 

Lab: 

1. Process LN tissue to isolate lymphocytes in a biosafety cabinet. Briefly, manually remove fat and connective tissue with a scalpel and forceps in a petri dish or tissue culture dish. Cut tissue into a few small pieces. Use forceps to transfer tissue to a Miltenyi gentleMACS C tube. Fill tube with R10 to 10 mL. Run tissue on appropriate gentleMACS program for lymph node tissue. Filter cellular suspension through a 70 µM cell strainer into a 50 mL conical, using the plunger of a syringe if necessary.  Rinse filter with R10. Count cells. Centrifuge cells at 700 x g for 5 minutes at RT with acceleration and deceleration set to 9. 
   • Other tissue of interest can be processed as usual. We have not found processing to affect detection of infused antibodies (including Ficoll and Percoll density gradient centrifugation).  Extended post collection culture (e.g. for ICS staining following stimulation) is also possible.
   • To freeze cells and analyze in the future: resuspend cells in cryopreservation medium to approximately 10 million cells/mL. Aliquot 1 mL/cryovial and freeze. 
   • For immediate staining and analysis: resuspending cells in PBS, spin at 1000 x g for 3 minutes, and remove supernatant. Transfer cells to FACS tubes or 96-well plate and continue. 
2. Make LIVE/DEAD viability stain
   • Reconstitute LIVE/DEAD viability stain with 50 µL DMSO. 
   • Dilute reconstituted LIVE/DEAD viability stain 1:40 in distilled water. 
   • Dilute again 1:20 in PBS for a final dilution of 1:800. 
   • Note: diluted viability stain cannot be saved and reused
3. Resuspend washed cells with 100 µL 1:800 viability stain/sample. Incubate, covered, at room temperature,, for 20 minutes. 
4. During the viability incubation, make ex vivo antibody mix with a staining volume of 100 µL/sample in staining buffer.
   • If using multiple polymer dye-conjugate antibodies (e.g., Brilliant Violet), include Super Bright Complete Staining Buffer in antibody cocktail. 
5. Wash cells with flow cytometry staining buffer.
   • For cells in 5 mL FACS tubes – add 3 mL staining buffer, spin at 863 x g for 3 minutes, remove supernatant.  
   • For cells in 96-well plate – add ~150 µL staining buffer to each well (for a total of ~250 µL/well), spin at 1000 x g for 3 minutes, remove supernatant. Repeat twice more for a total of 3 washes.
6. Resuspend cells in 100 µL ex vivo staining buffer. Incubate, covered, at RT, for 20 minutes. 
7. Wash as above. 
8. Resuspend cells in 250 µL 0.5% PFA (4% PFA diluted 1:8 in PBS). 
9. Make single stain compensation samples using cells or beads. 
10. Acquire cells on flow cytometer. 

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