Fig 1.
Flow chart of the thawing and staining process (on the left). Variations in the thawing procedure (on the right).
Fig 2.
Gating strategy to identify live/dead CD45+ leukocytes.
A) Events were triggered on FSC-H at a deliberately low threshold to avoid accidental exclusion of dead cells. B) Doublet exclusion C) Identification of CD45 positive cells D) Dead cells identified as 7-AAD positive events. Compensation for spectral overlap was not required.
Fig 3.
PBMC viability (A) and absolute recovery (B) as a result of varying thawing time in 37°C heated water bath.
Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.
Fig 4.
Performance of six different washing media presented in order of declining performance.
Values presented with mean +/- SD. (A) PBMC viability (%) and (B) live PBMCs by thawing medium. Paired samples from 20 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.
Fig 5.
Impact of washing-medium temperature on viability (A) and PBMC recovery (B).
20 donors included. Ref.: reference group. **: p<0.01.
Fig 6.
Comparing two ways of mixing PBMCs and washing medium by viability (A) and absolute count of live PBMCs (B).
Paired samples 20 donors included. Ref.: reference group. NS: non-significant. **: p<0.01.
Fig 7.
Temperature of centrifuge by PBMC viability (A) and absolute live PBMC recovery (B).
Paired samples from 20 donors included. Ref.: reference group. NS: non-significant.
Fig 8.
Centrifugation time and force and PBMC viability (A) and absolute live PBMC count (B).
Paired samples from 18 donors included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.
Fig 9.
PBMC viability (A) and absolute live PBMC recovery (B) by incubation.
Samples were stored in a 37°C incubator with 5% CO2. Paired samples from 20 donors were included. Ref.: reference group. NS: non-significant. *: p<0.05. **: p<0.01.