PBMC Isolation Protocol
This page documents the detailed PBMC isolation protocol used in the Russian Immune Diversity Atlas (RIDA_v1.0) study, including catalog numbers, lot numbers, centrifugation specifications, and reagents. This is the technical companion to the manuscript prose in Methods.
RIDA_v1.0 collected samples from four sites: Moscow, Kazan, Grozny, and Yakutsk. The protocol was harmonized across all sites.
Blood Collection
- Volume: 8 mL per participant
- Collection tube: CPT tubes with sodium heparin (BD Vacutainer CPT, cat. no. 362753)
- Processing window: within 2 hours of collection, kept at room temperature
Reagents
| Reagent | Supplier | Catalog No. | Lot No. |
|---|---|---|---|
| Fetal bovine serum | Sigma-Aldrich | F2442 | 19G014, 20A363 |
| ACK lysing buffer | Thermo Fisher Scientific | A10492 | — |
| PBS (pH 7.4) | — | — | — |
| EDTA | — | — | — |
| CryoStor CS10 | STEMCELL Technologies | 079555 | — |
Centrifugation Protocol
Step 1 — Density-gradient separation
- Rotor: Swing-out
- Speed: 1,500 × g
- Time: 30 min
- Temperature: 20 °C
- Settings: Reduced acceleration and deceleration
- Pre-spin: Invert tube 8–10 times
Step 2 — PBMC harvest wash
- Speed: 300 × g
- Time: 15 min
- Temperature: 20 °C
- After: Remove plasma, harvest PBMC layer
Step 3 — Erythrocyte lysis
- Reagent: ACK lysing buffer (Thermo Fisher Scientific, cat. no. A10492)
- Purpose: Remove residual erythrocytes from cell pellet
Step 4 — Wash steps (×2)
- Buffer: PBS (pH 7.4) + 1% FBS + 1 mM EDTA
- Speed: 300 × g
- Time: 15 min
- Temperature: 20 °C
Cryopreservation
- Freezing medium: CryoStor CS10 (STEMCELL Technologies, cat. no. 079555)
- Initial cooling: −80 °C overnight in controlled-rate freezing container
- Long-term storage: Liquid nitrogen
CryoStor Catalog Note
The draft key resources table lists CryoStor CS10 as STEMCELL Technologies Cat# 100-1061, while this protocol page lists Cat# 079555. These likely reflect different packaging sizes or regional SKUs of the same product.
Genetic Multiplexing and Sample Pooling
Thawing, washing, and pooling of PBMCs for genetic multiplexing were performed at the main study site in Moscow.
Thawing medium: RPMI (Gibco, cat. no. 21870076) + 5% human serum (Sigma-Aldrich, cat. no. H4522) + 1% penicillin–streptomycin (Gibco, cat. no. 15140122) + 1% L-glutamine (Gibco, cat. no. 25030081).
Wash medium: RPMI + 10% FBS + 1% penicillin–streptomycin + 1% L-glutamine.
Final wash buffer: PBS + 0.04% BSA (Capricorn Scientific, cat. no. BSA-1S).
Procedure:
- Rapid thaw in 37 °C water bath (1–2 min)
- Transfer to pre-warmed thawing medium
- Centrifuge at 300 × g for 5 min at 21 °C
- Wash once with pre-warmed wash medium
- Wash twice with pre-warmed PBS + 0.04% BSA
- Filter through 30 μm MACS SmartStrainer (Miltenyi Biotec)
- Count with trypan blue (1:1) on Countess II FL (Thermo Fisher)
- Resuspend at 1.5 × 10⁶ cells/mL in PBS + 0.04% BSA
- Pool equal cell numbers and volumes from individual donors per batch
- All manipulations on ice after washing
Notes
- FBS (Sigma F2442) was used during both the isolation procedure and subsequent pooling/washing steps
- Reduced acceleration and deceleration settings were applied during all centrifugation steps to minimize cell stress
- The protocol was harmonized across all participating sites