Isolation of Human Primary CD4+ T cells

Materials and Reagents

1.Tips
0.1-10 μl (Ucallm, catalog number: L1101)
2-200 μl (Ucallm, catalog number: L1103)
100-1,000 μl (Ucallm, catalog number: L1110) 
2.Pipettes
2 ml Disposable Pasteur pipettes (Ucallm, catalog number: J8212)
5 ml Serological pipettes (Ucallm, catalog number: J2205)
10 ml Serological pipettes (Ucallm, catalog number:J2210 )
25 ml Serological pipettes (Ucallm, catalog number: J2325)
3.50 ml conical tube (Ucallm, catalog number:M2050)
4.Blood or LRC
5.RosetteSepTM Human CD4+ T cell enrichment cocktail (STEMCELL Technologies, catalog number: 15062)
6.Histopaque 1077 (Sigma-Aldrich, catalog number: 10771-500ML)
7.Fetal bovine serum (FBS) (Gemini Bio-Products, BenchMarkTM, catalog number: 100-106)  
8.1x PBS (Corning, catalog number: 21-031-CV)
9.Ammonium chloride (NH₄Cl) (Sigma-Aldrich, catalog number: A9434-500G)
10.Potassium bicarbonate (KHCO₃) (Sigma-Aldrich, catalog number: 237205- 100G )
11.Ethylenediaminetetraacetic acid disodium salt dihydrate (Na₂EDTA) (Sigma-Aldrich, catalog number: E6635-100G )
12.AKC lysis buffer (see Recipes)

Recipes

1.AKC Lysis buffer
850 ml H₂O
8.02 g (150 mM) of NH₄Cl
1 g (10 mM) of KHCO₃
37.2 mg (0.1 mM) of Na₂EDTA
Adjust pH to 7.2
Add H₂O to 1,000 ml, and store at 4 °C for months

Equipment

1. 1. Pipetman
   2. Pipette-Aids
   3. Vortex (VWR, catalog number: 10153-838 )
   4. Tabletop centrifuge for 96-well plates, Eppendorf, 15 ml and 50 ml tubes(Beckman Coulter, model: Allegra X-14R )
   5. Hemacytometer (Hausser Scientific, catalog number: 3200 ) or automated cell counter (ORFLO Technologies, catalog number: MXZ001 ) with cassettes type M (ORFLO Technologies, catalog number: MXC001 )
   6. Biosafety cabinet ‘Level 2’

Procedure

Note: This protocol describes CD4+ T cells isolation using the RosetteSep Human CD4+ T Cell Enrichment Cocktail. Any other CD4+ T cells isolation kit can be used, but the protocol might slightly differ. Always follow the manufacturer’s protocol.

1. 1. Orderfresh blood or LRC in advance to have it delivered on the day of the  experiment.
   2. Transfer blood into a 50 ml conical tube(if using LRC, cut both extremities of  the chamber with clean scissors and let the blood drops into the tube).
   3. Add 1,800 μl of RosetteSep Cocktail to sample, incubate at RT for 20 min,and mix sample by swirling the tube every 5 min.
   4. In a new 50 ml conicaltube, add 10 ml of Histopaque-1077.
   5. Dilute sample with equal volume of PBScontaining 2% FBS.
   6. Slowly and carefully layer diluted sample on density gradient medium to minimize their mixing.
   7. Centrifuge sample for 20 min at 800 x g, RT, with brake off.
   8. CD4+ T cells are contained in the white ring, below the plasma phase. Pipetthe ring and transfer it to a new 50 ml conical tube. 
   9. Wash cells by filling up the tube with PBS containing 2% FBS.
   10. Centrifuge for 3 min at 800 x g and discard supernatant.
   11. If pellet appears red, resuspend pellet with 15 ml of cold AKC lysis buffer(see Recipes), incubate for 2 min at RT.
   12. Fill the tube up to 50 ml with PBS containing 2% FBS,centrifuge for 3 min at 800 x g, and get rid of the supernatant.
   13. If the pellet is still red, repeat Steps 11-12, if not, proceed to Step 14. Resuspend pellet (CD4+ T cells) with PBS containing 2% FBS.
   14. Count cells using an automated cell counter or a hemacytometer.

References

1. 1. Battivelli, E., Dahabieh, M. S., Abdel-Mohsen, M., Svensson, J. P., Tojal Da Silva, I., Cohn, L. B.,
   2. Battivelli, E. and Verdin, E. (2018). HIVGKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4+ T Cells. Bio-protocol 8(20): e3050. DOI: 10.21769/BioProtoc.3050.Gramatica, A., Deeks, S., Greene, W. C., Pillai, S. K. and Verdin, E. (2018). Distinct chromatin functional states correlate with HIV latency reactivation in infected primary CD4+ T cells. Elife 7: e34655.

Manufacturer’s Link

STEMCELL Technologies: https://www.stemcell.com
Sigma-Aldrich: https://www.sigmaaldrich.com
GeminiBio: https://www.geminibio.com
Corning: https://www.corning.com
VWR: https://www.vwr.com
Beckman Coulter: https://www.beckmancoulter.com
Hausser Scientific: http://www.hausserscientific.com
Orflo Technologies: https://www.orflo.com

Disclaimer:

If any infringement is identified, please contact 【info@ucallmlabs.com】for content removal.